Method of Treating Rheumatoid Arthritis With An Anti-IL-6R Antibody

ABSTRACT

The present invention provides methods of preventing or treating rheumatoid arthritis using a fully human antibody or antigen-binding fragment thereof that specifically binds human interleukin-6 receptor (hIL-6R). The methods of the present invention may include administration of a second therapeutic agent, such as one or more of a non-steroidal anti-inflammatory drug (NSAID), a glucocorticoid, a disease-modifying anti-rheumatic drug (DMARD), or a TNF-alpha antagonist, T-cell blocker, anti-CD20 antibody, an IL-1, JAK or IL-17 antagonist, or any combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/286,261 filed Nov. 1, 2011, which is a divisional of U.S. patentapplication Ser. No. 12/780,006 filed on May 14, 2010, now U.S. Pat. No.8,080,248, which is a continuation-in-part of U.S. patent applicationSer. No. 12/501,657 filed on Jul. 13, 2009, now U.S. Pat. No. 8,043,617,which is a divisional of U.S. patent application Ser. No. 11/809,482filed on Jun. 1, 2007, now U.S. Pat. No. 7,582,298, which claims thebenefit under 35 U.S.C. §119(e) of U.S. provisional application Nos.60/810,664 filed on Jun. 2, 2006; and 60/843,232 filed on Sep. 8, 2006.U.S. patent application Ser. No. 12/780,006 also claims the benefitunder 35 U.S.C. §119(e) of U.S. provisional application Nos. 61/181,749filed on May 28, 2009; 61/262,661 filed on Nov. 19, 2009; and 61/297,302filed on Jan. 22, 2010. The disclosures of all the foregoing are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic treatment ofrheumatoid arthritis. More specifically, the invention relates to theuse of interleukin-6 receptor (IL-6R) antagonists, such as anti-IL-6Rantibodies, to treat rheumatoid arthritis.

BACKGROUND

Rheumatoid arthritis (RA) is an autoimmune disease characterized bychronic inflammation of synovial tissue, leading to destruction of thejoint architecture. It is recognized that cytokines such as tumornecrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6)play a role in joint inflammation and cartilage damage observed in RA.IL-6 is a pleiotropic cytokine with biological effects on many celltypes. IL-6 is often regarded as being downstream of TNF or IL-1 ininflammatory cytokine cascades and may therefore represent a commonpathway factor in a wide range of inflammatory processes. Blockade ofIL-6 signaling therefore offers the potential to ameliorate multiplepathogenic features of RA and other inflammatory diseases.

Therapeutic methods using IL-6R antagonists are mentioned in U.S. Pat.Nos. 5,888,510; 6,723,319; and 2001/0001663. Exemplary anti-IL-6Rantibodies are described in U.S. Pat. Nos. 7,582,298; 6,410,691;5,817,790; 5,795,695; 6,670,373; and 7,582,298.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides human antibodies, preferablyrecombinant human antibodies, that specifically bind human interleukin-6receptor (hIL-6R). These antibodies are characterized by binding tohIL-6R with high affinity and slow dissociation kinetics and by theability to neutralize IL-6 activity. The antibodies can be full-length(for example, an IgG1 or IgG4 antibody) or may comprise only anantigen-binding portion (for example, a Fab, F(ab′)₂ or scFv fragment),and may be modified to effect functionality, e.g., to eliminate residualeffector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933). In apreferred embodiment, the invention provides an antibody orantigen-binding fragment thereof, which binds human IL-6 receptor (SEQID NO:1) with a K_(D) of about 500 pM or less, as measured by surfaceplasmon resonance. In a more specific embodiment, the antibody orantigen-binding fragment has a K_(D) of less than 300 pM, or less than200 pM, or even less than 100 pM. In various embodiments, the antibodyor antigen-binding fragment thereof blocks hIL-6 activity with an IC₅₀of 250 pM or less, as measured by luciferase bioassay. In more specificembodiments, the antibody or antigen-binding fragment thereof exhibitsan IC₅₀ of 150 pM or less.

In related aspects, the antibody or antigen-binding fragment of theinvention binds hIL-6R with an affinity at least 2-fold higher than itbinds monkey IL-6R. In more preferred embodiments, the antibody orantigen-binding fragment binds hIL-6R protein (SEQ ID NO:1) with anaffinity that is up to about 3-fold higher relative to its binding tomonkey IL-6R (Macaca fascicularis extracellular domain shown in SEQ IDNO:251).

In one embodiment, the antibody or antigen-binding portion of theantibody of the invention comprises a heavy chain variable region (HCVR)selected from the group consisting of SEQ ID NO:3, 227, 19, 231, 35, 51,67, 83, 99, 115, 131, 147, 239, 241, 163, 179, 235, 195 and 211, orsubstantially similar sequence thereof. In a more specific embodiment,the antibody or antigen-binding fragment thereof further comprises alight chain variable region (LCVR) selected from the group consisting ofSEQ ID NO: 11, 229, 27, 233, 43, 59, 75, 91, 107, 123, 139, 155, 171,187, 203 and 219, or a substantially similar sequence thereof. Inspecific embodiments, the antibody or antigen-binding fragment thereofcomprise HCVR/LCVR pairs selected from the group consisting of SEQ IDNO:3/11; 227/229; 19/27; 231/233; 35/43; 51/59; 67/75; 83/91; 99/107;115/123; 131/139; 147/155; 239/155; 241; 155; 163/171; 179/187; 235/237;195/203; and 211/219, or substantially similar sequences thereof.

In a second aspect, the invention provides isolated nucleic acidmolecules that encode an antibody or antigen-binding fragment of anantibody of the invention. In one embodiment, the nucleic acid moleculeof the invention encodes an antibody or fragment thereof comprising anHCVR as described above. In specific embodiments, the nucleic acidmolecule encoding the HCVR is selected from the group consisting of SEQID NO:2, 226, 18, 230, 34, 50, 66, 82, 98, 114, 130, 146, 238, 240, 162,178, 234, 194 and 210, or a substantially identical sequence thereof. Ina related aspect, the invention provides an isolated nucleic acidmolecule encoding an LCVR as described above. In specific embodiments,the nucleic acid molecule encoding the LCVR is a nucleotide sequenceselected from the group consisting of SEQ ID NO: 10, 228, 26, 232, 42,58, 74, 90, 106, 122, 138, 154, 170, 186, 236, 202 and 218, or asubstantially identical sequence thereof.

In a third aspect, the invention features an antibody or antigen-bindingfragment, comprising a heavy chain complementary determining region 3(CDR3) domain and a light chain CDR3 domain, wherein: the heavy chainCDR3 domain comprises an amino acid sequence of the formulaX¹—X²—X³—X⁴—X⁵X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—X¹⁸-X¹⁹ (SEQID NO:247) wherein X¹=Ala, X²=Lys, X³=Gly, X⁴=Arg, X⁵=Asp, X⁶=Ser orAla, X⁷=Phe, X⁸=Asp; X⁹=Ile, X¹⁰=Pro or absent, X¹¹=Phe or absent,X¹²=Val or absent, X¹³=Tyr or absent, X¹⁴=Tyr or absent, X¹⁵=Tyr orabsent, X¹⁶=Gly or absent, X¹⁷=Met or absent, X¹⁸=Asp or absent, andX¹⁹=Val or absent; and the light chain CDR3 domain comprises an aminoacid sequence of the formula X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹ (SEQ ID NO:250)wherein X¹=Gln, X²=Gln or His, X³=Ala, X⁴=Asn or Tyr, X⁵=Ser, X⁶=Phe,X⁷=Pro, X⁸=Pro, and X⁹=Thr.

In a more specific embodiment, the antibody or antigen-binding fragmentfurther comprises: a heavy chain CDR1 domain comprising an amino acidsequence of the formula X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸ (SEC) ID NO:245) whereinX¹=Gly or Arg, X²=Phe, X³=Thr, X⁴=Phe, X⁵=Asp, X⁶=Asp, X⁷=Tyr, andX⁸=Ala; a heavy chain CDR2 domain comprising an amino acid sequence ofthe formula X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸ (SEQ ID NO:246) wherein X¹=Ile orVal, X². Ser, X³=Trp, X⁴=Asn, X⁵=Ser, X⁶=Gly, X⁷=Ser, and X⁸=Ile; lightchain CDR1 domain comprising an amino acid sequence of the formulaX¹—X²—X³—X⁴—X⁵—X⁶ (SEQ ID NO:248), wherein X¹=Gln, X²=Gly, X³=Ile,X⁴=Ser, X⁵=Ser, and X°=Trp; and a light chain CDR2 domain comprising anamino acid sequence of the formula X¹-X²-X³ (SEQ ID NO:249), whereinX¹=Gly or Ala, X²=Ala, and X³. Ser.

In a fourth aspect, the invention features an antibody orantigen-binding fragment, comprising: a heavy chain CDR3 domain selectedfrom the group consisting of SEQ ID NO: 25, 153, 9, 185, 41, 57, 73, 89,105, 121, 137, 169, 201 and 217; and a light chain CDR3 domain selectedfrom the group consisting of SEQ ID NO:33, 161, 17, 193, 49, 65, 81, 97,113, 129, 145, 177, 209 and 225.

In a more specific embodiment, the antibody or antigen-binding fragmentfurther comprises: a heavy chain CDR1 domain selected from the groupconsisting of SEQ ID NO: 21, 149, 5, 181, 37, 53, 69, 85, 101, 117, 133,165, 197, and 213; a heavy chain CDR2 domain selected from the groupconsisting of SEQ ID NO: 23, 151, 7, 183, 39, 55, 71, 87, 103, 119, 135,167, 199 and 215; a light chain CDR1 domain selected from the groupconsisting of SEQ ID NO: 29, 157, 13, 189, 45, 61, 77, 93, 109, 125,141, 173, 205 and 221; and a light chain CDR2 domain selected from thegroup consisting of SEQ ID NO: 31, 159, 15, 191, 47, 63, 79, 95, 111,127, 143, 175, 207 and 223.

In specific embodiments, the antigen or antigen-binding fragmentcomprises heavy chain CDR sequences SEQ ID NO:21, 23, 25 and light chainCDR sequences SEQ ID NO:29, 31, 33; heavy chain CDR sequences SEQ IDNO:149, 151, 153 and light chain CDR sequences SEQ ID NO:157, 159, 161;heavy chain CDR sequences SEQ ID NO:5, 7, 9 and light chain SEQ ID NO:13, 15, 17; and heavy chain CDR sequences SEQ ID NO: 181. 183, 185 andlight chain CDR sequences SEQ ID NO:189, 191, 193.

In a fifth aspect, the invention features isolated nucleic acidmolecules encoding an antibody or antigen-binding fragments of theinvention, wherein the antibody or fragment thereof comprises: a heavychain CDR3 domain encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO:24, 152, 8, 184, 40, 56, 72, 88, 104, 120,136, 168, 200 and 216; and a light chain CDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:32,160, 16, 192, 48, 64, 80, 96, 112, 128, 144, 176, 208 and 224; as wellas substantially identical nucleic acid sequences thereof.

In a more specific embodiment, isolated nucleic acid molecules areprovided encoding an antibody or antigen-binding fragment of theinvention, wherein the antibody or fragment thereof comprises: a heavychain CDR1 encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:20, 148, 4, 180, 36, 52, 68, 84, 100, 116, 132,164, 196 and 212; a heavy chain CDR2 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:22, 150, 6,182, 38, 54, 70, 86, 102, 118, 134, 166, 198 and 214; a light chain CDR1domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:28, 156, 12, 188, 44, 60, 76, 92, 108, 124, 140,172, 204 and 220; and a light chain CDR2 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:30, 158, 14,190, 46, 62, 78, 94, 110, 126, 142, 174, 206 and 222; as well assubstantially identical nucleic acid sequences thereof.

The invention encompasses anti-hIL-6R antibodies or antigen-bindingfragments thereof having a modified glycosylation pattern. In someapplications, modification to remove undesirable glycosylation sites maybe useful, or an antibody lacking a fucose moiety on an oligosaccharidechain, for example, to increase antibody-dependent cellular cytotoxicity(ADCC) (see Shield et al. (2002) JBC 277:26733). In other applications,modification of a galactosylation can be made in order to modifycomplement-dependent cytotoxicity (CDC).

In further aspects, the invention provides recombinant expressionvectors carrying the nucleic acid molecules of the invention, and hostcells into which such vectors have been introduced, as are methods ofmaking the antibodies or antigen-binding fragments of the inventionobtained by culturing the host cells of the invention. The host cell maybe a prokaryotic or eukaryotic cell, preferably the host cell is an E.coli cell or a mammalian cell, such as a CHO cell.

In a further aspect, the invention features a pharmaceutical compositioncomprising a human antibody or antigen-binding fragment of an antibodywhich specifically binds hIL-6R and a pharmaceutically acceptablecarrier.

The present invention additionally provides methods for treatingrheumatoid arthritis. The methods of the present invention compriseadministering to a patient in need of such treatment a therapeuticallyeffective amount of a human antibody or antigen-binding fragment of anantibody which specifically binds to human interleukin-6 receptor(hIL-6R).

The studies summarized in Examples 8-12 below utilize an anti-hIL-6Rantibody referred to as “mAb1.” This antibody is also referred to hereinas VQ8F11-21. mAb1 (VQ8F11-21) comprises an HCVR/LCVR amino acidsequence pair having SEQ ID NOs:19127, andHCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3 domains represented by SEQ IDNOs:21-23-251 SEQ ID NOs:29-31-33. However, the methods of the presentinvention can be practiced using any anti-IL-6R antibody disclosedherein, as well as variants and antigen-binding fragments of suchantibody.

Examples 10-12 were designed to determine the effects of mAb1administration on inflammation, as well as the safety and tolerabilityof mAb1 in RA patients, and to determine the time course of bioeffect onRA-associated markers after a subcutaneous dose of mAb1. As demonstratedin Example 12, dose-dependent reduction in high-sensitivity C-reactiveprotein (hsCRP) was observed through day 15 (p<0.0047). Suppression ofserum amyloid A (SAA), erythrocyte sedimentation rate (ESR) and serumhepcidin was also observed in a dose-related manner. Significantincreases in IL-6 were also observed. At day 8, a 200 mg dose of mAb1was associated with median percent changes of −91.7% in hsCRP, -92.5% inSAA, -33.8% in ESR, -66.2% in hepcidin, and +647.0% in IL-6.

Safety data from all three studies (Examples 10-12) were combined [mAb1(n=71) or placebo (n=24)]. During a maximum 16-week exposure period,16.9% and 2.4% of patients receiving mAb1 and placebo had at least oneneutrophil count of 1.0−1.5×10³/uL; and 7.0% and 0% had a neutrophilcount of 0.5−1.0×10³/uL. During exposure, 50.1% and 20.1% of patientsreceiving mAb1 and placebo had at least one alanine aminotransferase(ALT) elevation 1-3 times the upper limit of normal (×ULN); 1.4% and4.2% had ALT 3-5×ULN; and 1.4% and 0% had ALT >5×ULN. No alterations inneutrophils or liver enzymes were associated with adverse clinicaloutcomes.

In summary, IL-6R inhibition with subcutaneous administration of mAb1was well tolerated in patients with RA with no dose-limiting toxicitiesobserved. Target blockade was demonstrated by the significant increasein IL-6 after treatment. mAb1 administered to active RA patientsresulted in dose-related reduction in hsCRP, SAA, and ESR; the observedreduction of hepcidin within one week of treatment is believed to be thefirst reported demonstration of hepcidin reduction in RA in humans.Moreover, hsCRP was suppressed for two weeks after a single 200 mg doseof mAb1, suggesting that weekly or bi-weekly SC dosing may prove to beefficacious.

The present invention also includes methods of modifying a rheumatoidarthritis-associated biomarker in a patient by administering to thepatient an anti-hIL-6R antibody or antigen-binding fragment thereof.Exemplary RA-associated biomarkers include, e.g., high-sensitivityC-reactive protein (hsCRP), serum amyloid A (SAA), erythrocytesedimentation rate (ESR), serum hepcidin, hemoglobin, and interleukin-6(IL-6).

According to certain aspects of the present invention, the anti-hIL-6Rantibody may be administered to a patient subcutaneously (s.c.) orintravenously (iv). The anti-hIL-6R antibody may also be administered tothe patient in combination with one or more additional therapeuticagents. In certain embodiments, the anti-hIL-6R antibody is administeredin multiple, sequential doses to a patient.

The present invention further includes the use of any of the anti-hIL-6Rantibodies, antigen-binding fragments, and/or pharmaceuticalformulations disclosed herein in the manufacture of a medicament for thetreatment, prevention and/or amelioration of rheumatoid arthritis.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99,3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference todescribe in their entirety.

Anti-hIL-6R Antibodies

The present invention includes methods that comprise administering to apatient a human antibody, or an antigen-binding fragment thereof, thatbinds specifically to hIL-6R. As used herein, the term “hIL-6R” means ahuman cytokine receptor that specifically binds human interleukin-6(IL-6). In certain embodiments, the antibody that is administered to thepatient binds specifically to the extracellular domain of hIL-6R. Theextracellular domain of hIL-6R is shown in the amino acid sequence ofSEQ ID NO:1.

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and (optionally)constant domains. Such DNA is known and/or is readily available from,e.g., commercial sources, DNA libraries (including, e.g., phage-antibodylibraries), or can be synthesized. The DNA may be sequenced andmanipulated chemically or by using molecular biology techniques, forexample, to arrange one or more variable and/or constant domains into asuitable configuration, or to introduce codons, create cysteineresidues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding fragment,” as usedherein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (1) V_(H)-C_(H2); (ii)V_(H)-C_(H2); (iii) V_(H)-C_(H3); (iv) V_(H)-C_(H1)-C_(H2), (v)V_(H)-C_(H1)-C_(H2)-C_(H3), (vi) V_(H)-C_(H2)-C_(H3), (vii) V_(H)-C_(L);(viii) V_(L)-C_(H1); (ix) V_(L)-C_(H2), (x) V_(L)-C_(H3); (xi)V_(L)-C_(H1)-C_(H2), (xii) V_(L)C_(H1)-C_(H2)-C_(H3); (xiii)V_(L)-C_(H2)-C_(H3); and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

The term “specifically binds,” means that an antibody or antigen-bindingfragment thereof forms a complex with an antigen that is relativelystable under physiologic conditions. Specific binding can becharacterized by a dissociation constant of at least about 1×10⁻⁶ M orsmaller. Methods for determining whether two molecules specifically bindare well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like.

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

In specific embodiments, the antibody or antibody fragment for use inthe method of the invention may be a multispecific antibody, which maybe specific for different epitopes of one target polypeptide or maycontain antigen-binding domains specific for epitopes of more than onetarget polypeptide. An exemplary bi-specific antibody format that can beused in the context of the present invention involves the use of a firstimmunoglobulin (Ig) C_(H3) domain and a second Ig C_(H3) domain, whereinthe first and second Ig C_(H3) domains differ from one another by atleast one amino acid, and wherein at least one amino acid differencereduces binding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference. In oneembodiment, the first Ig C_(H3) domain binds Protein A and the second IgC_(H3) domain contains a mutation that reduces or abolishes Protein Abinding such as an H95R modification (by IMGT axon numbering; H435R byEU numbering). The second C_(H3) may further comprise an Y96Fmodification (by IMGT; Y436F by EU). Further modifications that may befound within the second C_(H3) include: D16E, L18M, N44S, K52N, V57M,and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU)in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S,K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S,K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M,R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies.Variations on the bi-specific antibody format described above arecontemplated within the scope of the present invention.

A “neutralizing” or “blocking” antibody, as used herein, is intended torefer to an antibody whose binding to hIL-6R results in inhibition ofthe biological activity of hIL-6. This inhibition of the biologicalactivity of hIL-6 can be assessed by measuring one or more indicators ofhIL-6 biological activity known to the art, such as hIL-6-inducedcellular activation and hIL-6 binding to hIL-6R (see examples below).

The fully-human anti-IL-6R antibodies disclosed herein may comprise oneor more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy and light chain variabledomains as compared to, the corresponding germline sequences. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases. The present inventionincludes antibodies, and antigen-binding fragments thereof, which arederived from any of the amino acid sequences disclosed herein, whereinone or more amino acids within one or more framework and/or CDR regionsare back-mutated to the corresponding germline residue(s) or to aconservative amino acid substitution (natural or non-natural) of thecorresponding germline residue(s) (such sequence changes are referred toherein as “germline back-mutations”). A person of ordinary skill in theart, starting with the heavy and light chain variable region sequencesdisclosed herein, can easily produce numerous antibodies andantigen-binding fragments which comprise one or more individual germlineback-mutations or combinations thereof. In certain embodiments, all ofthe framework and/or CDR residues within the V_(H) and/or V_(L) domainsare mutated back to the germline sequence. In other embodiments, onlycertain residues are mutated back to the germline sequence, e.g., onlythe mutated residues found within the first 8 amino acids of FR1 orwithin the last 8 amino acids of FR4, or only the mutated residues foundwithin CDR1, CDR2 or CDR3. Furthermore, the antibodies of the presentinvention may contain any combination of two or more germlineback-mutations within the framework and/or CDR regions, i.e., whereincertain individual residues are mutated back to the germline sequencewhile certain other residues that differ from the germline sequence aremaintained. Once obtained, antibodies and antigen-binding fragments thatcontain one or more germline back-mutations can be easily tested for oneor more desired property such as, improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Antibodies and antigen-binding fragments obtainedin this general manner are encompassed within the present invention.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Epitopes may be either conformational or linear. Aconformational epitope is produced by spatially juxtaposed amino acidsfrom different segments of the linear polypeptide chain. A linearepitope is one produced by adjacent amino acid residues in a polypeptidechain. In certain circumstance, an epitope may include moieties ofsaccharides, phosphoryl groups, or sulfonyl groups on the antigen.

Therapeutic Administration and Formulations

The methods of the present invention comprise administering atherapeutically effective amount of an anti-hIL-6R antibody to apatient. As used herein, the phrase “therapeutically effective amount”means a dose of anti-hIL-6R antibody that results in a detectableimprovement in one or more symptoms associated with rheumatoid arthritisor which causes a biological effect (e.g., a decrease in the level of aparticular biomarker) that is correlated with the underlying pathologicmechanism(s) giving rise to the condition or symptom(s) of rheumatoidarthritis. For example, a dose of anti-hIL-6R antibody which causes animprovement in any of the following symptoms or conditions is deemed a“therapeutically effective amount”: chronic disease anemia, fever,depression, fatigue, rheumatoid nodules, vasculitis, neuropathy,scleritis, pericarditis, Felty's syndrome and/or joint destruction.

In accordance with the methods of the present invention, atherapeutically effective amount of anti-hIL-6R antibody that isadministered to the patient will vary depending upon the age and thesize (e.g., body weight or body surface area) of the patient as well asthe route of administration and other factors well known to those ofordinary skill in the art. In certain embodiments, the dose ofanti-hIL-6R antibody administered to the patient is from about 10 mg toabout 500 mg. For example, the present invention includes methodswherein about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg,about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg,about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg,about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg,about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg,about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg,about 190 mg, about 195 mg, about 200, about 205 mg, about 210 mg, about215 mg, about 220 mg, about 225 mg, about 230 mg, about 235 mg, about240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about290 mg, about 295 mg, about 300, about 325 mg, about 350 mg, about 375mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500mg, or more of anti-hIL-6R antibody is administered to the patient.

The amount of anti-hIL-6R antibody that is administered to the patientmay be expressed in terms of milligrams of antibody per kilogram ofpatient body weight (i.e., mg/kg). For example, the methods of thepresent invention include administering an anti-hIL-6R antibody to apatient at a daily dose of about 0.01 to about 100 mg/kg, about 0.1 toabout 50 mg/kg, or about 1 to about 10 mg/kg of patient body weight.

The methods of the present invention include administering multipledoses of an anti-hIL-6R antibody to a patient over a specified timecourse. For example, the anti-hIL-6R antibody can be administered about1 to 5 times per day, about 1 to 5 times per week, about 1 to 5 timesper month or about 1 to 5 times per year. In certain embodiments, themethods of the invention include administering a first dose ofanti-hIL-6R antibody to a patient at a first time point, followed byadministering at least a second dose of anti-hIL-6R antibody to thepatient at a second time point. The first and second doses, in certainembodiments, may contain the same amount of anti-hIL-6R antibody. Forinstance, the first and second doses may each contain about 10 mg toabout 500 mg, about 20 mg to about 300 mg, about 50 mg to about 200 mg,or about 75 mg to about 150 mg of the antibody. The time between thefirst and second doses may be from about a few hours to several weeks.For example, the second time point (i.e., the time when the second doseis administered) can be from about 1 hour to about 7 weeks after thefirst time point (i.e., the time when the first dose is administered).According to certain exemplary embodiments of the present invention, thesecond time point can be about 1 hour, about 4 hours, about 6 hours,about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 7days, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 14 weeks or longer after the first timepoint. Third and subsequent doses may be similarly administeredthroughout the course of treatment of the patient.

The invention provides methods of using therapeutic compositionscomprising anti-IL-6R antibodies or antigen-binding fragments thereof.The therapeutic compositions of the invention will be administered withsuitable carriers, excipients, and other agents that are incorporatedinto formulations to provide improved transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the weight of a subject tobe administered, target disease, conditions, route of administration,and the like. Various delivery systems are known and can be used toadminister the pharmaceutical composition of the invention, e.g.,encapsulation in liposomes, microparticles, microcapsules, receptormediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem.262:4429-4432). Methods of introduction include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, and oral routes. The composition may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local.

The pharmaceutical composition can also be delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533). Incertain situations, the pharmaceutical composition can be delivered in acontrolled release system, for example, with the use of a pump orpolymeric materials. In another embodiment, a controlled release systemcan be placed in proximity of the composition's target, thus requiringonly a fraction of the systemic dose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, localinjection, drip infusions, etc. These injectable preparations may beprepared by methods publicly known. For example, the injectablepreparations may be prepared, e.g., by dissolving, suspending oremulsifying the antibody or its salt described above in a sterileaqueous medium or an oily medium conventionally used for injections. Asthe aqueous medium for injections, there are, for example, physiologicalsaline, an isotonic solution containing glucose and other auxiliaryagents, etc., which may be used in combination with an appropriatesolubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol(e.g., propylene glycol, polyethylene glycol), a nonionic surfactant[e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct ofhydrogenated castor oil)], etc. As the oily medium, there are employed,e.g., sesame oil, soybean oil, etc., which may be used in combinationwith a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.The injection thus prepared is preferably filled in an appropriateampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the antibody contained isgenerally about 1 to 500 mg per dosage form in a unit dose; especiallyin the form of injection, it is preferred that the antibody is containedin about 5 to 100 mg and in about 10 to 250 mg for the other dosageforms.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

In accordance with the methods of the present invention, the anti-hIL-6Rantibody (or pharmaceutical formulation comprising the antibody) can beadministered to the patient using any acceptable device or mechanism.For example, the administration can be accomplished using a syringe andneedle or with a reusable pen and/or autoinjector delivery device. Themethods of the present invention include the use of numerous reusablepen and/or autoinjector delivery devices to administer an anti-hIL-6Rantibody (or pharmaceutical formulation comprising the antibody).Examples of such devices include, but are not limited to AUTOPEN™ (OwenMumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic MedicalSystems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen,HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I,II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (NovoNordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, FranklinLakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™(sanofi-aventis, Frankfurt, Germany), to name only a few. Examples ofdisposable pen and/or autoinjector delivery devices having applicationsin subcutaneous delivery of a pharmaceutical composition of the presentinvention include, but are not limited to the SOLOSTART™ pen(sanofi-aventis), the FLEXPEN™ (Nova Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.), to name only a few.

The use of a microinfusor to deliver an anti-hIL-6R antibody (orpharmaceutical formulation comprising the antibody) to a patient is alsocontemplated herein. As used herein, the term “microinfusor” means asubcutaneous delivery device designed to slowly administer large volumes(e.g., up to about 2.5 mL or more) of a therapeutic formulation over aprolonged period of time (e.g., about 10, 15, 20, 25, 30 or moreminutes). See, e.g., U.S. Pat. No. 6,629,949; U.S. Pat. No. 6,659,982;and Meehan et al., J. Controlled Release 46:107-116 (1996).Microinfusors are particularly useful for the delivery of large doses oftherapeutic proteins contained within high concentration (e.g., about100, 125, 150, 175, 200 or more mg/mL) and/or viscous solutions.

Combination Therapies

The present invention includes methods of treating rheumatoid arthritiswhich comprise administering to a patient in need of such treatment ananti-hIL-6R antibody in combination with at least one additionaltherapeutic agent. Examples of additional therapeutic agents which canbe administered in combination with an anti-hIL-6R antibody in thepractice of the methods of the present invention include, but are notlimited to NSAIDs, DMARDs, TNFα antagonists, T-cell blockers, CD-20antagonists (e.g., anti-CD-20 antibodies), IL-1 antagonists, JAKantagonists, IL-17 antagonists, and any other compound known to treat,prevent, or ameliorate rheumatoid arthritis in a human subject.Specific, non-limiting examples of additional therapeutic agents thatmay be administered in combination with an anti-hIL-6R antibody in thecontext of a method of the present invention include, but are notlimited to methotrexate, sulfasalazine, hydroxychloroquine, leflunomide,etanercept, infliximab, adalimumab, golimumab, rilonacept, anakinra,abatacept, certolizumab and rituximab. In the present methods, theadditional therapeutic agent(s) can be administered concurrently orsequentially with the anti-hIL-6R antibody. For example, for concurrentadministration, a pharmaceutical formulation can be made which containsboth an anti-hIL-6R antibody and at least one additional therapeuticagent. The amount of the additional therapeutic agent that isadministered in combination with the anti-hIL-6R antibody in thepractice of the methods of the present invention can be easilydetermined using routine methods known and readily available in the art.

Biomarkers

The present invention includes methods of treating rheumatoid arthritisby administering to a patient in need of such treatment atherapeutically effective amount of a human antibody or antibody bindingfragment thereof which specifically binds to hIL-6R, wherein the levelof one or more RA-associated biomarkers in the patient is modified(e.g., increased, decreased, etc., as the case may be) followingadministration. In a related aspect, the present invention includesmethods for decreasing an RA-associated biomarker in a patient byadministering to the patient a therapeutically-effective amount of ahuman antibody or antigen-binding fragment thereof which specificallybinds to hIL-6R.

Examples of RA-associated biomarkers include, but are not limited to,e.g., high-sensitivity C-reactive protein (hsCRP), serum amyloid A(SAA), erythrocyte sedimentation rate (ESR), serum hepcidin,interleukin-6 (IL-6), and hemoglobin (Hb). As will be appreciated by aperson of ordinary skill in the art, an increase or decrease in anRA-associated biomarker can be determined by comparing the level of thebiomarker measured in the patient at a defined time point afteradministration of the anti-IL-6R antibody to the level of the biomarkermeasured in the patient prior to the administration (i.e., the “baselinemeasurement”). The defined time point at which the biomarker can bemeasured can be, e.g., at about 4 hours, 8 hours, 12 hours, 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,15 days, 20 days, 35 days, 40 days or more after administration of theanti-hIL-6R antibody.

According to certain embodiments of the present invention, a patient mayexhibit a decrease in the level of one or more of hsCRP, SAA, ESR and/orhepcidin following administration of an anti-hIL-6R antibody to thepatient. For example, at about day 8 following administration of asingle dose of about 200 mg of an anti-hIL-6R antibody (e.g., mAb1), thepatient may exhibit one or more of the following: (i) a decrease inhsCRP by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or more; (ii) a decrease in SAA by about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more; (iii) a decrease in ESR byabout 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or more; and/or (iv) adecrease in hepcidin by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75% or more.

According to certain other embodiments of the present invention, apatient may exhibit an increase in the level of one or more of Hb orIL-6 following administration of an anti-hIL-6R antibody to the patient.For example, at about day 8 following administration of a single dose ofabout 200 mg of an anti-hIL-6R antibody (e.g., mAb1), the patient mayexhibit one or more of the following: (v) an increase in Hb by about0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%or more; and/or (vi) an increase in IL-6 by about 100%, 150%, 200%,250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%or more.

The present invention includes methods for determining whether a subjectis a suitable patient for whom administration of an anti-hIL-6R antibodywould be beneficial. For example, if an individual, prior to receivingan anti-hIL-6R antibody, exhibits a level of an RA-associated biomarkerwhich signifies the disease state, the individual is thereforeidentified as a suitable patient for whom administration of ananti-hIL-6R antibody would be beneficial. According to certain exemplaryembodiments, an individual may be identified as a good candidate foranti-hIL-6R therapy if the individual exhibits one or more of thefollowing: (i) a level of hsCRP greater than about 4 mg/L (e.g., about4.5 mg/L, about 5.0 mg/L, about 5.5 mg/L, about 6.0 mg/L, about 7.0mg/L, about 10.0 mg/L, about 15.0 mg/L, about 20.0 mg/L, or more); (ii)a level of SAA greater than about 3800 ng/mL (e.g., about 4000 ng/mL,4500 ng/mL, about 5000 ng/mL, about 5500 ng/mL, about 6000 ng/mL, about10,000 ng/mL, about 20,000 ng/mL, about 25,000 ng/mL, about 30,000ng/mL, about 35,000 ng/mL, about 40,000 ng/mL, about 45,000 ng/mL, ormore); (iii) an ESR greater than about 15 mm/hr (e.g., about 16 mm/hr,about 17 mm/hr, about 18 mm/hr, about 19 mm/hr, about 20 mm/hr, about 21mm/hr, about 22 mm/hr, about 25 mm/hr, about 30 mm/hr, about 35 mm/hr,about 40 mm/hr, about 45 mm/hr, about 50 mm/hr, or more); and/or (iv) alevel of hepcidin greater than about 60 ng/mL (e.g., about 62 ng/mL,about 64 ng/mL, about 68 ng/mL, about 70 ng/mL, about 72 ng/mL, about 74ng/mL, about 76 ng/mL, about 78 ng/mL, about 80 ng/mL, about 82 ng/mL,about 84 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, about100 ng/mL, about 105 ng/mL, or more). Additional criteria, such as otherclinical indicators of RA, may be used in combination with any of theforegoing RA-associated biomarkers to identify an individual as asuitable candidate for anti-hIL-6R therapy.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human IL-6 Receptor

Immunization of rodents can be done by any methods known in the art(see, for example, Harlow and Lane (1988) supra; Malik and Lillehoj,Antibody techniques: Academic Press, 1994, CA). In a preferredembodiment, hIL-6R antigen is administered directly to mice whichcomprise DNA loci encoding both human Ig heavy chain variable region andKappa light chain variable region (Veloclmmune™, RegeneronPharmaceuticals, Inc.; U.S. Pat. No. 6,596,541), with an adjuvant tostimulate the immune response. Such an adjuvant includes complete andincomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI(muramyl dipeptides) (see O'Hagan, Vaccine Adjuvant, by Human Press,2000, NJ). Such an adjuvant can prevent rapid dispersal of polypeptideby sequestering the antigen in a local depot, and may contain factorsthat can stimulate host immune response. In one embodiment, hIL-6R isadministered indirectly as DNA plasmid that contains hIL-6R gene andexpresses hIL-6R using the host cellular protein expression machinery toproduce antigen polypeptide in vivo. In both approaches, theimmunization schedule requires several administrations spaced by a fewweeks. The antibody immune response is monitored by standardantigen-specific immunoassay. When animals reached their maximum immuneresponse, the antibody expressing B cells were harvested and fused withmouse myeloma cells to preserve their viability, forming hybridomacells. To select functionally desirable monoclonal antibodies,conditioned media of the hybridoma cells or transfected cells werescreened for specificity, antigen-binding affinity, and potency inblocking hIL-6 binding to hIL-6R (described below).

Example 2 Anti-hIL6R Antibodies Generated Via Direct Isolation ofSplenocytes

DNA encoding VH and VL domains may be isolated directly from a singleantigen positive B cell. Briefly, the hIL-6Rα immunized transgenic mousewas terminated and splenocytes were harvested. Red blood cells wereremoved by lysis followed by pelleting the harvested splenocytes.Resuspended splenocytes were first incubated with a cocktail of humanIgG, FITC-anti-mFc, and biotin-IL6Ra for 1 hour. The stained cells werewashed twice with PBS, then stained with a cocktail of human and ratIgG, APC-anti-mIgM, and SA-PE for one hour. The stained cells werewashed once with PBS and were analyzed by flow cytometry on a MoFlo(Cytomation). Each IgG positive, IgM negative, and antigen positive Bcell was sorted and plated into a separate well on a 96-well plate.RT-PCR of antibody genes from these B cells was performed according to amethod described by Wang et al. (2000) (J Immunol Methods 244:217-225).Briefly, cDNAs for each single B cell were synthesized via RT-PCR. Eachresulting RT product was then split and transferred into twocorresponding wells on two 96-well plates. One set of the resulting RTproducts was first amplified by PCR using a 5′ degenerate primerspecific for human IgG heavy chain variable region leader sequence and a3′ primer specific for mouse heavy chain constant region, to form anamplicon. The amplicon was then amplified again by PCR using a 5′degenerate primer set specific for framework 1 of human IgG heavy chainvariable region sequence and a nested 3′ primer specific for mouse heavychain constant region. The other set of the resulting RT products wasfirst amplified by PCR using a 5′ degenerate primer specific for humankappa light chain variable region leader sequence and a 3′ primerspecific for mouse kappa light chain constant region to form anamplicon. The amplicon was then amplified again by PCR using a 5′degenerate primer set specific for framework 1 of human kappa lightchain variable region sequence and a nested 3′ primer specific for mousekappa light chain constant region. The heavy chain and light chain PCRproducts were cloned into Sap I-linearized antibody vectors containingIgG1 heavy chain constant region and kappa light chain constant region,respectively. The heavy chain plasmid has a lox2272 site and a lox511site flanking the heavy chain expression cassettes. In addition,immediately downstream of the lox2272 in the heavy chain plasmid thereis a hygromycin-resistance gene that lacks a promoter and an initiatingATG. The hygromycin-resistance gene is also transcriptionally linked toa downstream eGFP gene via an IRES sequence. The light chain plasmid hasa loxP site and lox2272 site flanking the light chain expressioncassette. In addition, The light chain plasmid has a SV40 promoterimmediately before an ATG at the lox2272 site, such that uponintegration into an appropriate host cell the lox2272-proximal SV40promoter and initiating ATG from the light chain plasmid is broughtadjacent to the hygromycin-resistance gene in the heavy chain plasmid inthe proper reading frame to allow transcription and translation of thehygromycin-resistance and eGFP genes. Purified recombinant plasmidshaving a heavy chain variable region sequence and plasmids having alight chain variable region sequence from the same B cell were thencombined and transfected, together with a plasmid that expresses the Crerecombinase, into a modified CHO host cell line. The modified CHO hostcell line contains, from 5′ to 3′, a loxP site, an eCFP, a lox2272 site,DsRed, and a lox511 site at a transcriptionally active locus.Consequently, the host CHO cell can be isolated by flow cytometry as ablue-positive, red-positive, and green-negative cell. When recombinantplasmids expressing heavy chain and light chain genes are transfectedtogether with a plasmid expressing the Cre recombinase, site-specificrecombination mediated by the Cre recombinase results in the integrationof the antibody plasmids at the chromosomal locus containing the loxsites and replacement of the eCFP and DsRed genes. Recombinants can thenbe isolated as blue-negative, red-negative, and green-positive cells byflow cytometry. Accordingly, CHO cells transfected with recombinantplasmids having a heavy chain variable region sequence and plasmidshaving a light chain variable region sequence from the same B cell weresorted by flow cytometry, and proper recombinants that show theblue-negative, red-negative, and green-positive phenotype were isolated,and stable recombinant antibody-expressing CHO cell lines wereestablished from isolated clones.

Example 3 Antigen Binding Affinity Determination

K_(D) of the antigen binding to the selected antibodies described abovewere determined by surface kinetics on a real-time biosensor surfaceplasmon resonance assay (BIAcorem). More specifically, the affinity ofthe antibodies for human IL-6R was measured using a BIAcore® 2000 orBIAcore® 3000. The antibody was captured on an anti-mouse IgG surfaceand exposed to various concentrations of recombinant hIL-6R proteineither in monomeric or dimeric form. Kinetic analysis usingBIAevaluation™ software was performed to obtain the association anddissociation rate constants.

Binding affinities of the antibodies to hIL-6R was also measured foreither hybridoma-conditioned media or purified proteins by plate-basedcompetition immunoassay. The antibody proteins were purified usingProtein G affinity chromatography from hybridoma cell conditioningmedium that was bovine IgG-depleted (Invitrogen). For the competitionELISA, briefly, constant amounts of antibody at different levels werepremixed with serial dilutions of antigen protein, hIL-6R-hFc, rangingfrom 0 to 10 μg/ml, and incubated for two hours at room temperature toreach pseudo-binding equilibrium between the antibody and antigen. Thesesolutions were then transferred to 96-well hIL-6R-hFc pre-coated platesto allow the free-antibody in the mixtures to bind to plate-coatedhIL-6R-hFc. The plates were typically coated with 1 to 2 μg/mlhIL-6R-hFc protein in PBS solution overnight at 4° C. followed by BSAnonspecific blocking. After washing off excess antibody in solution,plate-bound antibodies were detected with an HRP-conjugated goatanti-mouse IgG or IgA polyclonal antibody reagent and developed usingeither colorimetric or chemiluminescence substrates. The dependency ofthe signals on the concentrations of antigen in solution was analyzedwith a 4-parameter fit analysis using Prism™ software (Graph Pad) andreported as IC₅₀. Competition immunoassay were also carried out usingsteady state solution phase Kinexa™ instrument (Sapidyne Inc.).

Results are shown in Table 1 (control: humanized monoclonal antibody tohuman IL-6R (U.S. Pat. No. 5,817,790 SEQ ID NO:69 and 71). Antibody(HCVR and LCVR amino acid sequences): VQ8A9-6 (3, 11); VQ8F11-21 (19,27); VV7G4-1 (35, 43); VV7G4-10 (51, 59) VV6C10-1 (67, 75); VV6C10-3(83, 91); VV6C10-4 (99, 107); VV6F12-11 (115, 123); VV9A6-11 (131, 139);VV6A9-5 (147, 155), VV3D8-4 (163, 171); VV1G4-7 (179, 187);248982-13-1-E5 (195, 203); 248982-13-2-A9 (211, 219). Monomer and dimerK_(D) determined by BIAcore™; solution K_(D) by Kinexa™; IC₅₀ by ELISAassays (n.d.=not determined).

TABLE 1 Antigen Binding Affinity K_(D) K_(D) Solution K_(D) ELISA IC₅₀Monomer Dimer Monomer Dimer Antibody (nM) (nM) (nM) (nM) VQ8A9-6 0.2220.101 0.120 0.004 VQ8F11-21 0.067 0.023 0.009 0.008 VV3D8-4 2.410 0.1721.910 0.013 VV6A9-5 0.097 0.146 0.032 0.005 VV1G4-7 0.225 0.070 0.1970.041 VV6C10-1 0.267 0.032 2.050 0.010 VV6F12-11 n.d n.d n.d 0.033VV7G4-10 n.d. n.d. n.d. 1.980 VV9A6-11 n.d. n.d. n.d. 0.347 VV6C10-3n.d. n.d. n.d. 0.009 248982-13-1-E5 0.987 0.785 n.d. 0.360248982-13-2-A9 2.870 n.d. n.d. 0.054 Control 1.790 n.d. 1.960 n.d.

Example 4 Neutralization of hIL-6 Activity

hIL-6 blocking activities of the anti-hIL-6R antibodies of the inventionwere screened by hIL-6 blocking immunoassays, in vitro hIL-6 dependentcell growth bioassays, and surface plasmon resonance (BIAcore™). Theimmunoassay was used to screen ability of the tested antibody to blockhIL-6 binding to hIL-6R, and the in vitro bioassay was used to determinethe potency of the antibodies in neutralizing hIL-6R-mediated cellularsignal transduction.

For the immunoassay, hIL-6 recombinant protein was coated on a 96-wellplate in PBS buffer overnight at 4° C. This plate was used to capturefree hIL-6R-hFc from antibody sample solutions, and the amount ofcaptured hIL-6R-hFc was quantified according to the standard curve. Thesample solutions were composed of a constant amount of hIL-6R-hFcrecombinant protein (100 pM) and varying amounts of antibody, either incrude hybridoma condition medium or as purified antibody protein,ranging from 0 to about 50 nM in serial dilutions. The antibody-antigenmixtures were incubated at room temperature for ˜2 hours to allowantibody-antigen binding to reach equilibrium. The equilibrated samplesolutions were then transferred to the hIL-6 coated plates formeasurement of free hIL-6R-hFc. After 1 hour binding, the plate waswashed and bound hIL-6R-hFc was detected using HRP-conjugated goatanti-hFc polyclonal antibodies (Jackson Immuno Research), and developedusing TMB substrate (BD Pharmigen). IC₅₀s were determined as the amountof antibody required to reduce 50% of IL-6R-hFc detectable to platebound hIL-6 ligand. Results are shown in the first column of Table 2.

Additionally, the ability of the test antibody to block hIL-6 binding tothe hIL-6R receptor was determined using surface plasmon resonance.Purified antigen hIL-6R-hFc molecules were captured by goat anti-humanIgG polyclonal antibodies immobilized on CM-5 surface through aminecoupling to a density of 250 RU. hIL-6 solution (0.25 ml, 50 nM) wasinjected over the receptor surface and bound hIL-6 recorded (firstinjection of IL-6). Bound hIL-6 was then removed with a pulse of 3 MMgCl₂ following by conditioning buffer. Anti-hIL6R antibody in hybridomaconditioned medium was injected over the captured receptor surfacefollowed by second injection of hIL-6. The percent reduction in hL-6binding resulting from preformed antibody and receptor complex was usedas a score to define hIL-6 blockers from non-blockers (second column,Table 2).

TABLE 2 Neutralization of hIL-6 Binding hIL6R/hIL6 hIL6/hIL6R XG-1 cellHepG2/Stat3 Binding Binding proliferation Luciferase InhibitionInhibition Inhibition activity Antibody IC₅₀ (nM) (%) IC₅₀ (nM) IC₅₀(nM) VQ8A9-6 0.39 68 0.40 0.097 VQ8F11-21 0.12 98 0.62 0.135 VV3D8-40.61 93 >100 n.d. VV6A9-5 0.35 100 1.10 0.188 VV1G4-7 1.10 34 1.80 0.578VV6C10-1 4.60 61 >6.90 n.d. VV6F12-11 2.20 n.d. n.d. n.d. VV7G4-10 13.00n.d. n.d. n.d. VV9A6-11 0.50 n.d. n.d. n.d. VV6C10-3 0.06 n.d. n.d. n.d.Control 2.20 91 1.50 0.854

The ability of hIL-6R antibodies to block hIL-6 activity in vitro wasmeasured in the hIL-6-dependent myeloma line XG-1. XG-1 cells maintainedin hIL-6-containing medium were washed twice with hIL-6-free media andcultured for ˜24 hours in hIL-6-free medium to deplete residual hIL-6.The starved cells were then spun down and re-suspended in the medium at4×10⁵ cells per ml and plated 20,000 cells per well in a 96-well tissueculture plate. The purified antibody proteins were serially diluted inmedium and added to the plated cells at concentrations ranging from 0 to50 nM. Subsequently, recombinant hIL-6 was added to the wells to a finalconcentration of 8 pM. Cells were allowed to grow for ˜72 hours at 37°C. in a humidified 5% CO₂ incubator. At the end of growth period, livecells were measured using CCK-8 kit (Dojindo, Japan). IC₅₀s weredetermined as described above, and reported in the third column of Table2.

The ability of hIL-6R antibodies to block hIL-6 activity was alsomeasured in vitro in the hIL-6-responsive human hepatoma cell line,HepG2. HepG2 cells were transfected with a reporter plasmid containing aSTATS (Signal Transducer and activator of Transcription 3) responseelement linked to a luciferase gene. The transfected cells weretrypsinized, spun down and re-suspended in the medium at approximately2.5×10⁵ cells per ml and plated at 20,000 cells per well in a 96-welltissue culture plate. The purified antibody proteins were seriallydiluted in medium and added to the plated cells at concentrationsranging from 0 to 100 nM. Subsequently, recombinant hIL-6 was added tothe wells to a final concentration of 50 pM. The response was measuredafter incubating the cells for 6 hours at 37° C. in a humidified 5% CO₂incubator. Luciferase activity was measured with the Steady-Glo™luciferase assay system (Promega). IC₅₀s were determined as describedabove, and reported in the fourth column of Table 2.

Example 5 Binding Epitope Diversity

An antibody binding competition immunoassay was performed using as acontrol humanized antibody to human IL-6R. Briefly, a 96-wellimmunosorbent plate was coated with 20 ng per well hIL-6R recombinantprotein overnight at 4° C. After blocking non-specific binding with BSA,the hIL-6R binding sites on one half of the plate were saturated withbinding of the control antibody by addition of 500 ng of the control perwell, and to the other half of the plate was added binding buffer only.After three hours binding at room temperature, the purified antibodieswere spiked in at a final concentration of 50 ng/ml with and without thepreexisting control antibody in the well. After one hour of additionalbinding, the free antibody was washed away and the plate-bound antibodywas detected with HRP-conjugated goat anti-mouse IgG or IgA, polyclonalantibody and the plate was developed using chromatic HRP substrates andabsorbance at 450 nm was recorded. Percentage deductions of the bindingof the anti-hIL6R antibodies by the presence of the control antibody arelisted in Table 3 below. A similar experiment was conducted usingsurface plasmon resonance technology (Table 3). Both methods generatedconsistent results. Antibodies VQ8F11, VV3D8, VV6A9, VV6C10-1 boundepitopes overlapping with the control antibody; while antibodies VQ8A9,VV1G4, VV6F12, VV7G4, VV9A6, and VV6C10-3 appeared to bind distinctepitopes as antigen binding was not blocked by the control antibody.Partial competition may result from steric hindrance from the firstantibody bound, even though epitopes may not be overlapping.

TABLE 3 Competition of Antigen Binding with Control Antibody BIAcore ™Immunoassay Antibody (% reduction) (% reduction) VQ8A9-6 26 3 VQ8F11-2196 79 VV3D8-4 97 84 VV6A9-5 96 84 VV1G4-7 12 3 VV6C10-1 90 80 VV6F12-11n.d. 3 VV7G4-10 n.d. 26 VV9A6-11 n.d. 18 VV6C10-3 n.d. 1

Example 6 Cross-Species Binding Property

Four antibodies were tested for cross-reactivity to monkey IL-6Rrecombinant protein using BIAcore™ technology. Briefly, a biosensor chipon which goat anti-mouse Fc polyclonal antibody was immobilized was usedto present anti-hIL-6R monoclonal antibodies to a density of about 75RU. Recombinant human or monkey monomeric IL-6R protein (Macacafascicularis, extracellular domain; SEQ ID NO:251), at a concentrationrange between 1.25-40 nM, was injected over the antibody surface. Thebinding of the receptor to the antibody and the dissociation of thebound complex were monitored in real-time. Both association rateconstant (ka) and dissociate rate constant (kd) were obtained, and K_(D)calculated (Table 4).

TABLE 4 Comparison of Binding Affinity to Human and Monkey IL-6RAntibody Antigen ka (M⁻¹S⁻¹) kd (S⁻¹) K_(D) (nM) Control Human IL6R1.74E+05 1.67E−04 0.963 Monkey IL6R 1.44E+05 1.68E−04 1.170 VQ8F11-21Human IL6R 8.51E+05 4.38E−05 0.051 (mAb1) monkey IL6R 3.39E+05 4.86E−050.143 VV1G4-7 Human IL6R 2.57E+05 6.18E−05 0.240 monkey IL6R no bindingVV6A9-5 Human IL6R 5.18E+05 8.41E−05 0.162 monkey IL6R 5.00E+05 7.70E−050.154 VQ8A9-6 Human IL6R 7.32E+05 2.76E−04 0.377 monkey IL6R 7.31E+054.16E−04 0.569

Among the four tested antibodies, VQ8F11, VV6A9, and VQ8A9 stronglyreacted to monkey receptor with K_(D) values that differed by about 1.5-to about 3-fold from human receptor binding, respectively. VV1G4, whichwas not blocked by the control antibody (Table 3), showed no binding tomonkey receptor despite strong binding to the human receptor with K_(D)of 241 pM.

Example 7 Effect of Constant Region on Binding Affinity

The binding affinity to monomeric hIL-6R of four antibodies having mouseIgG, human IgG1 or human IgG4 (wild-type and modified) were determinedusing BIAcore™ as described above except a goat anti-human Fc polyclonalantibody surface was used to capture hIgG antibodies. Monomeric hIL-6Rwas injected at concentrations of 12.5, 6.25, 3.12, and 1.56 nM. Theability of the antibodies to neutralize hIL-6-dependent HepG2/STAT3signal transduction was also determined in a luciferase assay (IC₅₀).IC₅₀s for different IgG isotypes were similar, suggesting no effect ofisotype on antibody affinity for antigen.

TABLE 5 Comparison of IgG Isotypes Antibody IgG ka (M⁻¹S⁻¹) kd (S⁻¹)K_(D) (nM) IC₅₀ (nM) VQ8F11-21 hIgG1 6.22E+05 4.54E−05 0.073 0.150(mAb1) hIgG4 7.17E+05 5.22E−05 0.073 0.228 mIgG2a 7.86E+05 5.27E−050.067 0.135 modhIgG4 8.81E+05 4.705−05 0.053 0.249 VQ8A9-6 hIgG11.09E+06 2.60E−04 0.238 0.130 hIgG4 1.17E+06 2.35E−04 0.201 0.185 mIgG19.95E+05 2.21E−04 0.222 0.097 VV6A9-5 hIgG1 7.12E+05 8.87E−05 0.1250.204 hIgG4 5.67E+05 7.64E−05 0.135 0.343 mIgG2a 7.72E+05 7.52E−05 0.0970.188 VQ1G4-21 hIgG1 3.34E+05 7.92E−05 0.237 0.767 hIgG4 2.73E+059.18E−05 0.336 0.528 mIgG2a 3.41E+05 7.66E−05 0.225 0.578

Example 8 Pharmacokinetics Studies of a Human Anti-IL-6R Antibody mAb1

The pharmacokinetics of mAb1 (HCVR SEQ ID NO:19 and LCVR SEQ ID NO:27)was examined in cynomolgus monkeys following a single subcutaneous (SC)or IV injection at multiple dose levels. Doses for SC administrationwere 1, 5, and 15 mg/kg; IV doses were 1 and 15 mg/kg. Blood sampleswere collected from all animals (N=6 per group, 3 per sex per group) atselected time points over a 52 day (1248 hr) time course. The resultantserum samples were analyzed using a validated ELISA assay for total mAb1concentrations. The data were analyzed by means of noncompartmentalmethods. Pharmacokinetic (PK) parameter estimates such as observedmaximal concentration in serum (C_(max)), the time of observed maximalconcentration (T_(max)), area under the concentration vs. time curve(AUC), clearance (CL), volume of distribution (Vz), and mean residencetime (MRT) were determined. The bioavailability following subcutaneousadministration was approximately 78%.

At low doses, mAb1 had a half-life of 28 to 30 hr. At high doses, theterminal half-life estimate was approximately 225 hr when drug levelswere above 10 μg/ml. The half-life was approximately 80 hr when serumconcentrations were below 10 μg/ml.

Example 9 Toxicology Studies of Anti-IL-6R Antibody mAb1

In a GLP toxicology study, mAb1 was administered to cynomolgus monkeysvia SC injection twice weekly for 13 consecutive weeks (total of 26doses). Groups of 12 animals (6/sex) were administered 0 (placebo), 1,5, 15 or 50 mg/kg/dose. Four animals/sex scheduled for the primarynecropsy within 1 week of the end of dosing period and the remaining 2animals/sex/group were assigned to a 12-week nondosing recovery period.The animals were observed for mortality and moribundity. Clinicalexaminations were performed daily and detailed physical examinationswere performed weekly. Individual body weights were recorded weekly.Clinical pathology evaluations included hematology, serum chemistry,urine and CRP analysis. Blood samples were collected periodically fortoxicokinetic and antibody evaluation. Ophthalmic and electrocardiogramexaminations were performed periodically throughout the study. Completenecropsies were performed on all animals. Selected organs were weighedand selected tissues were examined microscopically. All animals survivedto the scheduled primary or recovery necropsies. There were no testarticle-related clinical findings or effects on appetite, body weights,ophthalmologic examination results, electrocardiographic parameters,macroscopic findings or organ weights.

Results indicated that administration of mAb1 via subcutaneous injectionto cynomolgus monkeys twice weekly for 13 consecutive weeks was welltolerated. Slight decreases in neutrophil counts, fibrinogen, and CRPvalues in monkeys administered mAb1 were considered to be effects orpossible effects of test article administration, but were not consideredto be adverse and in general, alterations in neutrophil counts andfibrinogen values resolved during the recovery period. CRP values werequite variable and alterations and subsequent resolution when comparedto controls were not consistent in all groups. Test article treatmentresulted in minimal to moderate perivascular mixed inflammatory cellinfiltrates in dermis and/or subcutis in subcutaneous injection sites.Full or partial reversibility was evident following the recovery period.Findings of unclear relationship to test article treatment includedsevere diffuse subacute inflammation in heart in a single femaleadministered 5 mg/kg and minimal focal subacute inflammation accompaniedby mild perivascular mononuclear cell infiltrates in the brain of asingle male administered 1 mg/kg. Toxicokinetic data indicatesubstantial systemic mAb1 exposure during the dosing phase of the study.In addition, 10 of the 16 recovery animals had circulating levels ofmAb1 throughout the entire recovery period, while 6 animals had nodetectable mAb1 at the end of the study. The no-observed-adverse-effectlevel (NOAEL) for subcutaneous injection of mAb1 to cynomolgus monkeystwice weekly for 13 consecutive weeks was 50 mg/kg.

Example 10 Single Dose Study in Subjects with Rheumatoid Arthritis

In order to evaluate the potential of mAb1 for treatment of RA, a studywas conducted to evaluate safety, tolerability and pharmacokinetics ofSC administered mAb1. The primary objective was to assess the safety andtolerability of a single dose of subcutaneously administered mAb1 insubjects with rheumatoid arthritis who were concomitantly treated withmethotrexate. The secondary objective was to assess the PK profile of asingle subcutaneous dose of mAb1 and immunogenicity of a single SC doseof mAb1

Endpoints. The primary efficacy endpoint was the percent change fromBaseline in hs-C reactive protein (“hs-CRP”). Exploratory endpoints werethe percent change in Subject's Assessment of Pain and Subject's GlobalAssessment of Disease activity.

Study Design. This is a multi-centered, randomized, double blind,placebo-controlled, single dose parallel group study of the safety,tolerability and pharmacodynamics of subcutaneously administered mAb1 insubjects with rheumatoid arthritis who were receiving concomitantmethotrexate. Three (3) sequential cohorts of 5 subjects (4:1active:placebo) were dosed SC with 50, 100, or 200 mg mAb1 or placebo.In each cohort, 1 week safety data from the first 2 subjects dosed wasreviewed prior to dosing of the remaining subjects in the cohort.Screening took place within the window of 2 weeks to 3 days prior to thestart of dosing (Day-14 to Day-3). On Study Day-1, the subjectsunderwent pre-dose study procedures and randomization. On Study Day 1,the subjects received SC blinded study drug or placebo. Subjectsreturned home following the 8 hour blood draw. Subjects returned to theclinic for outpatient visits on Study Days 3, 4, 8 (Week 1), 11, 15(Week 2), 22 (Week 3), 29 (Week 4), 43 (Week 6), 57 (Week 8), 85 (Week12) and 113 (Week 16), for safety assessments and blood sampling.Subjects completed an End of Study (EOS) visit on Study Day 113 (Week16). Subjects who completed the study participated in 14 study visits.

Subject Eligibility. Inclusion Criteria: 1. Male or female 18 years ofage; 2. Subjects must weigh >50 kg and <100 kg; 3. Diagnosis ofRheumatoid Arthritis (RA) as defined by the 1987 revised AmericanCollege of Rheumatology (ACR) criteria with disease duration of no lessthan 6 months and ACR class I-III; 4. Subjects must receive a minimum of8 weeks treatment with methotrexate (MIX) prior to the Screening visit.Subjects must be on a stable dose of MTX (7.5 to 25 mg/week) for aminimum of 4 weeks prior to the Screening Visit; 5. All subjects willtake folic acid 1 mg daily or 5 mg weekly with the MTX dose, to minimizetoxicity, according to local guidelines; 6. Oral prednisone 510 mg/dayis allowed, as long as the dose is stable for 4 weeks prior to Screeningand for the duration of the study; 7. For women of childbearingpotential, a negative serum pregnancy test at the Screening Visit(Visit 1) and a negative urine pregnancy test at Day-1; 8. For men andwomen of childbearing potential, willingness to utilize adequatecontraception and not become pregnant (or have their partner[s] becomepregnant) during the full course of the study. Adequate contraceptivemeasures include oral contraceptives (stable use for 2 or more cyclesprior to the Screening visit); IUD; DEPO-PROVERA®; NORPLANT® Systemimplants; bilateral tubal ligation; vasectomy; condom or diaphragm pluseither contraceptive sponge, foam or jelly.

Exclusion Criteria: 1. A history of Listeriosis or active tuberculosis(TB); 2. Persistent chronic or active recurring infection requiringtreatment with antibiotics, antivirals, or antifungals within 4 weeksprior to the Screening Visit; 3. History of prior articular orprosthetic joint infection; 4. History of a hypersensitivity reaction,other than localized injection site reaction (ISR), to any biologicalmolecule; 5. History of a hypersensitivity reaction to doxycycline,tetracycline or related compounds; 6. Significant concomitant illnesssuch as, but not limited to cardiac, renal, neurological,endocrinological, metabolic or lymphatic disease that would adverselyaffect the subject's participation in this study; 7. Uncontrolleddiabetes, defined as Hemoglobin A1c (HbA1c) ≧9.0% at the ScreeningVisit; 8. Presence of any of the following laboratory abnormalities atthe Screening Visit: WBC <4,000/μl; platelet count <150,000/μl;neutrophils <2000/μl, AST/ALT >1.5×ULN; 9. Serum creatinine ≧1.5×ULN atthe Screening Visit; 10. Subjects with a positive intradermal skintuberculin test (PPD 5TU)≧5 mm induration read at 48 to 72 hours afterplacement; 11. Chest radiograph (at the Screening visit) consistent withprior tuberculosis infection including, but not limited to, apicalscarring, apical fibrosis, or multiple calcified granulomata. This doesnot include non-caseating granulomata; 12. Use of parenteral orintra-articular glucocorticoids within 4 weeks prior to the ScreeningVisit; 13. Treatment with anakinra within two weeks prior to theScreening Visit; 14. Treatment with etanercept, cyclosporine,mycophenolate, tacrolimus, gold, penicillamine, sulfasalazine, orhydroxychloroquine within 4 weeks prior to the Screening Visit; 15.Treatment with adalimumab within 6 weeks prior to the Screening Visit;16. Treatment with abatacept, azathioprine, cyclophosphamide orinfliximab within 12 weeks prior to the Screening Visit; 17. Treatmentwith leflunomide or rituximab within 6 months prior to the ScreeningVisit; 18. Treatment with tocilizumab or any anti-IL-6 medications priorto Screening Visit; 19. Received administration of any live (attenuated)vaccine within 3 months prior to the Screening Visit; 20. Known historyof Human Immunodeficiency Virus (HIV) antibody; and/or positiveHepatitis B surface antigen (HBsAg), and/or positive Hepatitis Cantibody (HCV) at the Screening Visit; 21. History of malignancy otherthan carcinoma in-situ of the cervix, or adequately treated,non-metastatic squamous or basal cell carcinoma of the skin within fiveyears prior to Screening Visit; 22, History of demyelinating disease ormultiple sclerosis; 23. History of myeloproliferative disorder; 24.History of alcohol or drug abuse within the 5 years prior to theScreening Visit; 25. Any subject who has had surgery within 4 weeksprior to the Screening Visit; 26. Any subjects with planned electivesurgery; 27. Any other arthritic or medical condition that in theopinion of the investigator could interfere with study evaluations; 28.Participation in any clinical research study evaluating anotherinvestigational drug or therapy within 30 days or at least 5 half-lives,whichever is longer, of the investigational drug, prior to the ScreeningVisit.

Study Drug Dosage and Administration. Subjects returned to the clinicfor the Baseline visit on Day-1. Once eligibility was confirmed via predose procedures, the subject was randomly allocated to receive eithermAb1 or placebo. Dose assignments were determined by the allocationschedule. Dosing took place starting at approximately 08:00 on Day 1.Subjects were required to be fasting (no food or water) beginning atmidnight on Day-1. Study doses of 50, 100, and 200 mg were administeredvia subcutaneous injection. Each dose was administered in a singleinjection. All study drug injections were administered in the abdomen.

Dose Preparation. Study drug was supplied as lyophilized powder insterile, single-use vials. Each vial contained 250 mg of mAb1 andprovided a stock solution of 100 mg/ml after reconstitution. Placebo wassupplied in matched vials. mAb1 was reconstituted in Sterile Water ForInjection (WFI), and contained a withdrawable volume of up to 2.0 ml.Dosing volume was 0.5 ml for the 50 mg dose, 1.0 ml for the 100 mg dose,and 2.0 ml for the 200 mg dose.

Study Procedures and Visits. Physical Examination. A physicalexamination was conducted at the Screening visit, Day-1 (Visit 2), Day15 (Visit 7), and Day 113 (Visit 14, EOS). Vital Signs Vital signsincluding temperature, sitting blood pressure, pulse and respirationwere collected at every study visit. On Day 1 (Visit 3), vital signswere done prior to each pharmacokinetic blood draw at hour 0 (pre-dose)and at hour 8 post dose. Pharmacokinetic and Antibody Sample CollectionSerum samples were collected for pharmacokinetic (PK) analysis at everystudy visit beginning on Day 1 (Visit 3). On Day 1 (Visit 3), sampleswere collected at hour 0 (pre-dose) and at hour 8±3 minutes (post-dose).PK samples were subsequently collected at the same time each day onStudy Days 3, 4, 8, 11, 15, 22, 29, 43, 57, 85 and 113 (±2 hours). Serumsamples were collected for analysis of antibodies to mAb1 .

PPD Skin Test. Tuberculin purified protein derivative (PPD) 5TU skintest were placed intradermally at the time of the Screening visit andread 48 to 72 hours after inoculation. All subjects, with the exceptionof subjects who tested PPD positive and were successfully treated withanti-tuberculosis therapy, but including those with a prior history ofBacillus Calmette Guerin (BCG) administration received a PPD 5TU skintest. Subjects' successful treatment for a prior tuberculosis infectionwas documented in the source document, if applicable. Those subjectswith a positive PPD 5TU skin test, mm induration at 48 to 72 hours wereexcluded from the study.

Chest X-Ray. A radiologist's interpretation (signed and dated) of thestandard posterior-anterior and lateral chest X-rays noted the absenceof calcified granulomas and/or pleural scarring consistent with TB. Thisinformation was documented in the subject's medical chart and on theappropriate case report form at the Screening visit. A normal chestX-ray report was deemed acceptable if it had been done within the threemonths prior to the Screening visit.

Electrocardiogram. A standard 12-lead electrocardiogram (ECG) wasperformed at the Screening visit, Day 3 (Visit 4), Day 8 (Visit 6), Day15 (Visit 8) and Day 29 (Visit 10). Heart rate was recorded from theventricular rate and the PR, QRS, QT and QTc (QTc=QT/[60/heart rate]1/2)intervals were recorded.

Subject's Assessment of Pain. An 11-point scale (0=no pain to 10=severepain) was used to measure the subject's current level of pain. Thesubject was instructed to circle a box on the continuum indicating theappropriate response.

Subject's Global Assessment of Disease Activity. An 11-point scale (0=nosymptoms to 10=severe symptoms) was used to measure the subject'soverall assessment of his/her current disease activity. The subject wasinstructed to circle a box on the continuum indicating the appropriateresponse.

Additional Sample Collection. Blood and urine were collected asindicated for routine laboratory measurements. Subjects fasted beginningat midnight on the Day-1 (Visit 2, Baseline) and Day 43 (Visit 10, EOS)study visits. Plasma and/or serum samples were collected as indicatedand used for future analysis of serum proteins (i.e. proteomic analysis)as related to underlying disease in response to IL-6.

Schedule of Study Visits. Visit 1; Screening Visit (Day-14 to Day-3):Informed Consent, Inclusion/Exclusion Criteria, Medical History,Physical Examination, Height and Weight, Vital Signs, Chest x-ray, PPDSkin Test 5TU (read at 48 to 72 hours), Electrocardiogram, Serum HCGpregnancy test (for women of childbearing potential), Hepatitis C Virus(HCV) Ab, Hepatitis B Surface Antigen (HBsAg), HgbA1c, Hematology panel,Chemistry panel, Urinalysis, High sensitivity C-Reactive Protein(hs-CRP) and Serum Amyloid A (SAA), Erythrocyte Sedimentation Rate(ESR), IL-6 and fibrinogen, Concomitant medications, Adverse Events.Visit 2; Day-1: Vital Signs, Weight, Urine pregnancy test (for women ofchildbearing potential), Hematology panel, Chemistry panel, Urinalysis,Concomitant medications, Adverse events, Randomization. Visit 3, Dosing,Day 1, Baseline: Vital signs, Urine pregnancy test (for women ofchildbearing potential), Hematology panel, Chemistry panel, Urinalysis,hs-CRP and SAA, ESR, IL-6 and fibrinogen, RheumatoidFactor/ANA/anti-dsDNA, Serum immunoglobulins, Serum ferritin, Iron,Total Iron Binding Capacity (TIBC), RNA, Proteomics sample,Pharmacokinetic blood draw (0 hours pre-dose and 8 hours post dose),Anti-mAb1 antibody, Subject's Assessment of Pain, Subject's GlobalAssessment of Disease Activity, Concomitant medications, Adverse events.Visit 4, Day 3: Vital signs, Electrocardiogram, Hematology panel,Chemistry panel, Urinalysis, hs-CRP and SAA, ESR, IL-6 and fibrinogen,RNA, Proteomics sample, Pharmacokinetic blood draw, Anti-mAb1 antibody,Concomitant medications, Adverse events. Visit 5, Day 4: Vital signs,Hematology panel, Chemistry panel, Urinalysis, Pharmacokinetic blooddraw, RNA, Proteomic sample, Concomitant medications, Adverse events.Visit 6, Day 8: Vital signs, Electrocardiogram, Urine pregnancy test,Hematology panel, Chemistry panel, Urinalysis, hs-CRP and SAA, ESR, IL-6and fibrinogen, Pharmacokinetic blood draw, RNA, Proteomic sample,Subject's Assessment of Pain; Subject's Global Assessment of DiseaseActivity; Concomitant medications, Adverse events. Visit 7, Day 11:Vital signs, Hematology panel, Chemistry panel, Pharmacokinetic blooddraw, Concomitant medications, Adverse events. Visit 8, Day 15±1 day:Physical examination, Vital signs, Electrocardiogram, Urine pregnancytest, Hematology panel, Chemistry panel, Urinalysis, hs-CRP and SAA,ESR, IL-6 and fibrinogen, Pharmacokinetic blood draw, RNA, Proteomicssample, Subject's Assessment of Pain, Subject's Global Assessment ofDisease Activity, Concomitant medications, Adverse events. Visit 9, Day22±1 day: Vital signs, Hematology panel, Chemistry panel, Urinalysis,hs-CRP and SAA, ESR, IL-6 and fibrinogen, Pharmacokinetic blood draw,Concomitant medications, Adverse events. Visit 10, Day 29±1 day: Vitalsigns, Electrocardiogram, Urine pregnancy test, Hematology panel,Chemistry panel, Urinalysis, hs-CRP and SAA, ESR, IL-6 and fibrinogen,Serum ferritin, Iron, TIBC, Pharmacokinetic blood draw, RNA, Proteomicssample, Anti-mAb1 antibody, Subject's Assessment of Pain, Subject'sGlobal Assessment of Disease Activity, Concomitant medications, Adverseevents. Visit 11, Day 43±1 day: Vital signs, Hematology panel, Chemistrypanel, Subject's Assessment of Pain, Subject's Global Assessment ofDisease Activity, Pharmacokinetic blood draw, Concomitant medications,Adverse events. Visit 12, Day 57±1 day: Vital signs, Urine pregnancytest, Hematology panel, Chemistry panel, Urinalysis, hs-CRP and SAA,ESR, IL-6 and fibrinogen, Rheumatoid Factor/ANA/anti-dsDNA, Serumimmunoglobulins, Serum ferritin, Iron, TIBC, Pharmacokinetic blood draw,RNA, Proteomics sample, Anti-mAb1 antibody, Subject's Assessment ofPain, Subject's Global Assessment of Disease Activity, Concomitantmedications, Adverse events.

Visit 13, Day 85±1 day: Vital signs, Hematology panel, Chemistry panel,hs-CRP and SAA, ESR, IL-6 and fibrinogen, Pharmacokinetic blood draw,Concomitant medications, Adverse events. Visit 14, End of Study, Day113±3 day: Physical examination, Vital signs, Weight, Height, Urinepregnancy test, Electrocardiogram, Serum pregnancy test, Hematologypanel, Chemistry panel, Urinalysis, hs-CRP and SAA, ESR, IL-6 andfibrinogen, Rheumatoid Factor/ANA/anti-dsDNA, Serum immunoglobulins,Serum ferritin, Iron, TIBC, Pharmacokinetic blood draw, RNA, Proteomicssample, Anti-mAb1 antibody, Subject's Assessment of Pain, Subject'sGlobal Assessment of Disease Activity, Concomitant medications, Adverseevents.

Results: Baseline levels of RA-associated biomarkers (hsCRP, SAA, ESR,IL-6, Hb, and hepcidin) measured prior to administration of mAb1 orplacebo are shown in Table 6 (n=15).

TABLE 6 hsCRP 5.6 mg/L SAA 4252 ng/mL ESR 19 mm/hr IL-6 4.1 pg/mL Hb12.3 g/dL Mean hepcidin 82.5 ng/mL Median hepcidin 60.8 ng/mL

Median hepcidin levels (in ng/mL) over the course of the study are shownin Table 7.

TABLE 7 mAb1 dose (mg) Study Day 0 50 100 200 D1  57.6 38.5 66.3 87.8D4  8.8 39.6 22.8 49.3 D8  13.9 49.5 25.9 38.1 D29 0 24.0 55.5 66.5

The hepcidin levels for individual study participants is set forth inTable 8.

TABLE 8 Serum Hepcidin (ng/mL) Subject Treatment Day 1 Day 4 Day 8 Day29 1002  50 mg mAb1 30.39 38.57 43.99 0 1004  50 mg mAb1 229.22 40.55140.58 200.43 1005  50 mg mAb1 0 0 0 8.99 1003  50 mg mAb1 46.69 67.8355.09 39.00 1016 100 mg mAb1 8.01 0 25.35 0 1019 100 mg mAb1 71.82 45.5526.35 39.70 1020 100 mg mAb1 60.80 0 17.50 147.81 1017 100 mg mAb1 88.8849.77 156.34 71.36 1033 200 mg mAb1 0 9.00 0 0 1034 200 mg mAb1 37.1054.81 6.48 27.14 1031 200 mg mAb1 138.56 43.80 69.65 105.91 1032 200 mgmAb1 226.18 101.92 150.01 157.49 1001 placebo 0 0 0 0 1018 placebo115.12 229.96 169.06 96.82 1035 placebo 184.70 17.64 27.69 0

Safety was assessed by measuring neutrophils and alanineaminotransferase (ALT), as shown in Tables 9 and 10, respectively(ULN=upper limit of normal).

TABLE 9 Treatment Group 50 mg 100 mg 200 mg Combined mAb1 + mAb1 +mAb1 + mAb1 + Neutrophil MTX MTX MTX MTX MTX Range (n = 3) (n = 4) (n =4) (n = 4) (n = 12) <1.5 × 10³/μL 0 1 (25%) 0 2 (50%) 3 (25%) <1.0 ×10³/μL 0 0 0 0 0 <0.5 × 10³/μL 0 0 0 0 0

TABLE 10 Treatment Group 50 mg 100 mg 200 mg Combined mAb1 + mAb1 +mAb1 + mAb1 + MTX MTX MTX MTX MTX ALT Range (n = 3) (n = 4) (n = 4) (n =4) (n = 12) >1 × ULN 0 0 1 (25%) 3 (75%) 4 (33.3%) >2 × ULN 0 0 0 2(50%) 2 (16.7%) >3 × ULN 0 0 0 0 0 >5 × ULN 0 0 0 0 0 >8 × ULN 0 0 0 0 0

Example 11 Study in RA Subjects Receiving Concomitant Methotrexate

A second study was conducted to assess the safety and tolerability ofmultiple doses of subcutaneously administered mAb1 in subjects withrheumatoid arthritis who were receiving concomitant treatment withmethotrexate. The study was conducted in three parts and included atotal of 6 dose cohorts. Parts B and C began after the safety of Part Awas assessed.

Part A: Dose cohort 1: 10 subjects were randomized (4:1) to receiveeither: 50 mg mAb1 SC every week (8 subjects) or placebo every week (2subjects). Dose cohort 2: 10 subjects were randomized (4:1) to receiveeither: 100 mg mAb1 SC alternating with placebo every week (8 subjects),or placebo every week (2 subjects). Upon confirmation of the safety ofPart A, enrollment in Part B was opened: Part B: Dose cohort 3: 10subjects were randomized (4:1) to receive either: 100 mg mAb1 SC everyweek (8 subjects) or placebo every week (2 subjects). Dose cohort 4: 10subjects were randomized (4:1) to receive either: 200 mg mAb1 SCalternating with placebo every week (8 subjects), or placebo every week(2 subjects). Dose cohort 5: 10 subjects were randomized (4:1) toreceive either: 150 mg mAb1 SC every week (8 subjects) or placebo everyweek (2 subjects). Part C: Dose cohort 6: 10 subjects were randomized(4:1) to receive either: 150 mg mAb1 SC every other week (8 subjects) orplacebo every other week (2 subjects).

All subjects completed 5 weeks of treatment (dosing on Day 1, Weeks 1,2, 3 and 4), followed by 5 weeks of safety follow-up, for a totalduration of 10 weeks. Subjects completed 12 study visits (Screening, Day1 (first dose, Baseline Visit), Day 8 (Week 1), Day 15 (Week 2), Day 22(Week 3), Day 29 (Week 4), Day 36 (Week 5), Day 43 (Week 6), Day 50(Week 7), Day 57 (Week 8), Day 64 (Week 9) and Day 71 (Week 10).

Inclusion Criteria: 1. Male or female ≧18 years of age; 2. Subjectsweigh >50 kg and <100 kg; 3. Diagnosis of Rheumatoid Arthritis (RA) asdefined by the 1987 revised American College of Rheumatology (ACR)criteria with disease duration of no less than 6 months and ACR classI-III; 4. Subjects must have received a minimum of 12 weeks treatmentwith methotrexate (MTX) prior to the Screening visit. Subjects must beon a stable dose of MTX (7.5 to 25 mg/week) for a minimum of 6 weeksprior to the Screening Visit; 5. All subjects took folic acid 1 mg dailyor 5 mg weekly with the MTX dose, to minimize toxicity, according tolocal guidelines; 6. For men and women of childbearing potential,willingness to utilize adequate contraception and not become pregnant(or have their partner[s] become pregnant) during the full course of thestudy. Adequate contraceptive measures include oral contraceptives(stable use for 2 or more cycles prior to the Screening visit); IUD;DEPO-PROVERA®; NORPLANT® System implants; bilateral tubal ligation;vasectomy; condom or diaphragm plus either contraceptive sponge, foam orjelly.

Exclusion Criteria: 1. A history of Listeriosis or active tuberculosis(TB); 2. Persistent chronic or active recurring infection requiringtreatment with antibiotics, antivirals, or antifungals within 4 weeksprior to the Screening Visit, or any active infection at the time ofscreening or randomization; 3. History of prior articular or prostheticjoint infection; 4. History of a hypersensitivity reaction, other thanlocalized injection site reaction (ISR), to any biological molecule; 5.History of a hypersensitivity reaction to doxycycline, tetracycline orrelated compounds; 6. Significant concomitant illness such as, but notlimited to cardiac, renal, neurological, endocrinological, metabolic orlymphatic disease that would adversely affect the subject'sparticipation in this study; 7. Uncontrolled diabetes, defined asHemoglobin A1c (HbA1c) ≧9.0% at the Screening Visit; 8. Presence of anyof the following laboratory abnormalities at the Screening Visit: WBC<4,000/·L; platelet count <150,000·L; neutrophils <2000/·L,AST/ALT >1.5×ULN; 9. Serum creatinine×ULN at the Screening Visit; 10.Subjects with a positive intradermal skin tuberculin test (PPD 5TU)≧5 mminduration read at 48 to 72 hours after placement; 11. Chest radiograph(at the Screening visit) consistent with prior tuberculosis infectionincluding, but not limited to, apical scarring, apical fibrosis, ormultiple calcified granulomata. This does not include non-caseatinggranulomata; 12. Use of oral prednisone or equivalent >10 mg per daywithin 4 weeks prior to the Screening Visit; 13. Use of parenteral orintra-articular glucocorticoids within 4 weeks prior to the ScreeningVisit; 14. Treatment with anakinra within two weeks prior to theScreening Visit; 15. Treatment with etanercept, cyclosporine,mycophenolate, tacrolimus, gold, penicillamine, sulfasalazine, orhydroxychloroquine within 4 weeks prior to the Screening Visit; 16.Treatment with adalimumab within 6 weeks prior to the Screening Visit;17. Treatment with abatacept, azathioprine, cyclophosphamide orinfliximab within 12 weeks prior to the Screening Visit; 18. Treatmentwith leflunomide or rituximab within 6 months prior to the ScreeningVisit; 19. Use of tocilizumab or any other anti-IL-6 medication prior tothe Screening Visit; 20. Prior exposure to mAb1; 21. Receivedadministration of any live (attenuated) vaccine within 3 months prior tothe Screening Visit; 22. Known history of Human Immunodeficiency Virus(HIV) antibody; and/or positive Hepatitis B surface antigen (HBsAg),and/or positive Hepatitis C antibody (HCV) at the Screening Visit; 23.History of malignancy other than adequately treated carcinoma in-situ ofthe cervix, or adequately treated, non-metastatic squamous or basal cellcarcinoma of the skin within five years prior to Screening Visit; 24.History of demyelinating disease or multiple sclerosis; 25. History ofmyeloproliferative disorder; 26. History of alcohol or drug abuse withinthe 5 years prior to the Screening Visit; 27. Any subject who has hadsurgery within 4 weeks prior to the Screening Visit; 28. Any subjectswith planned elective surgery; 29. Any other arthritic or medicalcondition that in the opinion of the investigator could interfere withstudy evaluations; 30. Participation in any clinical research studyevaluating another investigational drug or therapy within 30 days or atleast 5 half-lives, whichever is longer, of the investigational drug,prior to the Screening Visit.

Study drug was supplied as a lyophilized powder in sterile, single-usevials. Each vial contained 269 mg of mAb1 and provided a stock solutionof 100 mg/mL after reconstitution. Placebo was supplied in matchedvials. mAb1 was reconstituted in Sterile Water for Injection (WFI), andcontained a withdrawable volume of up to 2 mL.

Study Drug Dosage. Subjects had a Screening Visit on Day-14 to Day-3.Once eligibility was confirmed, the subjects were randomly allocated toreceive either mAb1 or placebo. Dose assignment into each group wasdetermined by an IVRS. In Part A, subjects were randomized into two dosecohorts. Subjects in dose cohort 1 received either 50 mg mAb1 SC everyweek or matching placebo every week and subjects in cohort 2 receivedeither 100 mg mAb1 SC alternating with placebo every week or matchingplacebo every week. Subjects in dose cohort 2 received a dose of 100 mgSC mAb1 on Day 1 (Visit 2), Day 15 (Visit 4) and Day 29 (Visit 6) and adose of placebo on Day 8 (Visit 3) and Day 22 (Visit 5). Escalation toPart B took place after all 20 subjects in Part A completed the Day 36(Week 5) visit and the laboratory and safety data were reviewed, and thesafety of the 200 mg single dose was confirmed. Subjects in Part B wererandomized into three dose cohorts. Subjects in dose cohort 3 receivedeither 100 mg mAb1 SC every week or matching placebo every week;subjects in cohort 4 received either 200 mg mAb1 SC alternating withplacebo every week or matching placebo every week and subjects in dosecohort 5 received either 150 mg mAb1 SC every week or matching placeboevery week. Subjects in dose cohort 4 received a dose of 200 mg SC mAb1on Day 1 (Visit 2), Day 15 (Visit 4) and Day 29 (Visit 6) and a dose ofplacebo on Day 8 (Visit 3) and Day 22 (Visit 5).

Schedule of Study Visits. Screening; Visit 1; Day-14 to Day-3: InformedConsent; Inclusion/Exclusion Criteria; Medical History; PhysicalExamination; Height and Weight; Vital Signs; Chest x-ray (PA andLateral); PPD skin test 5 TU (to be read at 48 to 72 hours); hs-CRP;Complement (C3, C4 and CHSO); SAA, fibrinogen, IL-6; ESR;Electrocardiogram; Serum βHCG pregnancy test (for women of childbearingpotential); HCV Ab and HBsAg; HbA1c; Hematology panel; Chemistry panel;Urinalysis; Concomitant medications; Adverse Events. Visit 2; BaselineVisit; Day 1: Vital Signs, Weight; Urine pregnancy test (for women ofchildbearing potential); Hematology panel; Chemistry panel; Urinalysis;hs-CRP; Complement (C3, C4 and CHSO); SAA, fibrinogen, IL-6; ESR; Serumimmunoglobulins; Rheumatoid Factor/ANA/anti-dsDNA; RNA; Proteomicsample; Plasma for MTX analysis; Pharmacokinetic blood draw; Anti-mAb1antibody; Subject's Assessment of Pain; Subject's Global Assessment ofDisease Activity; Concomitant medications; Adverse events;Randomization; Study drug administration. Visit 3; Day 8 (Week 1) (±1day): Vital signs; Hematology panel; Chemistry panel; Urinalysis;hs-CRP; Complement (C3, C4 and CHSO); SAA, fibrinogen, IL-6; ESR; RNA;Proteomic sample; Plasma for MTX analysis; Pharmacokinetic blood draw;Concomitant medications; Adverse events; Study drug administration.Visit 4; Day 15 (Week 2) (±1 day): Vital signs; Hematology panel;Chemistry panel; Urinalysis; hs-CRP; Complement (C3, C4 and CHSO); SAA,fibrinogen, IL-6; ESR; Plasma for MTX analysis; Pharmacokinetic blooddraw; Concomitant medications; Adverse events; Study drugadministration. Visit 5: Day 22 (Week 3) (±1 day): Vital signs;Hematology panel; Chemistry panel; Urinalysis; hs-CRP; Complement (C3,C4 and CHSO); SAA, fibrinogen, IL-6; ESR; Plasma for MTX analysis;Pharmacokinetic blood draw; Concomitant medications; Adverse events;Study drug administration. Visit 6: Day 29 (Week 4) (±1 day): Vitalsigns; Hematology panel; Chemistry panel; Urinalysis; hs-CRP; Complement(C3, C4 and CHSO); SAA, fibrinogen, IL-6; ESR; Plasma for MTX analysis;Pharmacokinetic blood draw; Concomitant medications; Adverse events;Study drug administration. Visit 7: Day 36 (Week 5) (±1 day): Vitalsigns; Electrocardiogram; Urine pregnancy test (for women ofchildbearing potential); Hematology panel; Chemistry panel; Urinalysis;hs-CRP; Complement (C3, C4 and CHSO); SAA, fibrinogen, IL-6; ESR; Plasmafor MTX analysis; Pharmacokinetic blood draw; Anti-mAb1 antibody;Proteomic sample; RNA; Subject's Assessment of Pain; Subject's GlobalAssessment of Disease Activity; Concomitant medications; Adverse events.Visit 8; Day 43 (Week 6) (±1 day): Vital signs; Hematology panel;Chemistry panel; Urinalysis; hs-CRP; Complement (C3, C4 and CHSO);Plasma for MIX analysis; Pharmacokinetic blood draw; Concomitantmedications; Adverse events. Visit 9; Day 50 (Week 7) (±1 day): Vitalsigns, Hematology panel; Chemistry panel; Urinalysis; hs-CRP; Complement(C3, C4 and CHSO); Plasma for MTX analysis; Pharmacokinetic blood draw;Concomitant medications; Adverse events. Visit 10: Day 57 (Week 8) (±1day): Vital signs; Hematology panel; Chemistry panel; Urinalysis;hs-CRP; Complement (C3, C4 and CHSO); SAA, fibrinogen, IL-6; ESR; RNA;Proteomic sample; Plasma for MTX analysis; Pharmacokinetic blood draw;Concomitant medications; Adverse events. Visit 11: Day 64 (Week 9) (±1day): Vital signs; Hematology panel; Chemistry panel; Urinalysis;hs-CRP; Complement (C3, C4 and CHSO); Plasma for MTX analysis;Pharmacokinetic blood draw; Concomitant medications; Adverse events.Visit 12: Day 71 (Week 10) (±1 day); End of Study: Physical Examination;Vital Signs; Weight; Height; Electrocardiogram; Serum pregnancy test(for women of childbearing potential); Hematology panel; Chemistrypanel; Urinalysis; hs-CRP; Complement (C3, C4 and CHSO); SAA,fibrinogen, IL-6; ESR; Serum immunoglobulins; RheumatoidFactor/ANA/anti-dsDNA; RNA; Proteomic sample; Plasma for MTX analysis;Pharmacokinetic blood draw; Anti-mAb1 antibody; Subject's Assessment ofPain; Subject's Global Assessment of Disease Activity; Concomitantmedications; Adverse events.

Results: Baseline levels of RA-associated biomarkers (hsCRP, SAA, ESR,IL-6, Hb, and hepcidin) measured prior to administration of mAb1 orplacebo are shown in Table 11 (n=47).

TABLE 11 hsCRP 4.6 SAA 5800 ESR 25 IL-6 4.5 Hemoglobin 13.3 Meanhepcidin 102.6 Median hepcidin 76.9

Safety was assessed by measuring neutrophils and alanineaminotransferase (ALT), as shown in Tables 12 and 13, respectively(qw=weekly dosing; q2w=biweekly dosing of mAb1 alternating withplacebo).

TABLE 12 Treatment Group I Neutrophil 50 mg mAb1 qw + 100 mg mAb1 100 mgmAb1 Range MTX (n = 13) MTX (n = 8) q2w + MTX (n = 8) qw + MTX (n = 8)<1.5 × 10³/μL 1 (8%) 0 0 2 (25%) <1.0 × 10³/μL 0 0 0 1 (13%) <0.5 ×10³/μL 0 0 0 0 Treatment Group II Neutrophil 200 mg mAb1 150 mg mAb1 150mg mAb1 Combined mAb1 + Range q2w + MTX (n = 7) qw + MTX (n = 8) q2w +MTX (n = 8) MIX (n = 47) <1.5 × 10³/μL 3 (43%) 3 (38%) 0 8 (17%) <1.0 ×10³/μL 1 (14%) 1 (13%) 0 3 (6%) <0.5 × 10³/μL 0 0 0 0

TABLE 13 Treatment Group I 50 mg mAb1 qw + 100 mg mAb1 100 mg mAb1 ALTRange MTX (n = 13) MTX (n = 8) q2w + MTX (n = 8) qw + MTX (n = 8)   >1 ×ULN 3 (23 %) 2 (25%) 4 (50%) 6 (75%) >1.5 × ULN 0 1 (13%) 1 (13%) 4(50%)   >2 × ULN 0 0 0 2 (25%)   >3 × ULN 0 0 0 0 Treatment Group II 200mg mAb1 150 mg mAb1 150 mg mAb1 Combined mAb1 + ALT Range q2w + MTX (n =7) qw + MTX (n = 8) q2w + MTX (n = 8) MTX (n = 47)   >1 × ULN 4 (57%) 4(50%) 1 (13%) 21 (45%) >1.5 × ULN 2 (29%) 2 (25%) 0 10 (21%)   >2 × ULN1 (14%) 1 (13%) 0 4 (9%)   >3 × ULN 0 0 0 0

Example 12 Subcutaneously Administered mAb1 in Subjects with RheumatoidArthritis

A third study was conducted to assess the bioeffect of a single dose ofmAb1 compared with placebo in subjects with active rheumatoid arthritiswho were receiving concomitant treatment with methotrexate.

Study Design. The study was designed as a single-dose, double-blind,placebo-controlled, parallel group safety, tolerability andpharmacodynamic study of subcutaneously (SC) administered mAb1 inrheumatoid arthritis patients who are receiving concomitantmethotrexate. Four (4) parallel groups of 8 subjects each with activerheumatoid arthritis were dosed SC with 50, 100 or 200 mg mAb1 orplacebo (1:1:1:1). Each subject received a single dose of mAb1 orplacebo, and was followed for 6 weeks. Subjects (32) completed 10 studyvisits: (Screening, Day 1, Day 4, Day 8, Day 12, Day 15, Day 22, Day 29,Day 36 and Day 43).

Inclusion Criteria: 1. Male or female ≧18 years of age; 2. Subjects mustweigh >50 kg and <100 kg; 3. Diagnosis of Rheumatoid Arthritis (RA) asdefined by the 1987 revised American College of Rheumatology (ACR)criteria with disease duration of no less than 6 months and ACR classI-III; 4. Subjects must receive a minimum of 12 weeks treatment withmethotrexate (MTX) prior to the Screening visit. Subjects must be on astable dose of MTX (7.5 to 25 mg/week) for a minimum of 8 weeks prior tothe Screening Visit; 5. All subjects will take folic acid at 5 mg weeklyor greater with the MTX dose, to minimize toxicity; 6. hs-CRP 10 mg/L;7. For men and women of childbearing potential, willingness to utilizeadequate contraception and not become pregnant (or have their partner[s]become pregnant) during the full course of the study. Adequatecontraceptive measures include oral contraceptives (stable use for 2 ormore cycles prior to screening) and other prescription pharmaceuticalcontraceptives; IUD; bilateral tubal ligation; vasectomy; condom ordiaphragm plus either contraceptive sponge, foam or jelly.

Exclusion Criteria: 1. A history of Listeriosis or active tuberculosis(TB); 2. Persistent chronic or active recurring infection requiringtreatment with antibiotics, antivirals, or antifungals within 4 weeksprior to the Screening Visit; 3. History of prior articular orprosthetic joint infection; 4. History of a hypersensitivity reaction,other than localized injection site reaction (ISR), to any biologicalmolecule; 5. History of a hypersensitivity reaction to doxycycline,tetracycline or related compounds; 6. Significant concomitant illnesssuch as, but not limited to cardiac, renal, neurological,endocrinological, metabolic or lymphatic disease that would adverselyaffect the subject's participation in this study; 7. Uncontrolleddiabetes, defined as Hemoglobin A1c (HbA1c) ≧9.0% at the ScreeningVisit; 8. Presence of any of the following laboratory abnormalities atthe Screening Visit: WBC <4,000/μ; platelet <150,000/μl; neutrophils<2000/0, AST/ALT >1.5×ULN; 9. Serum creatinine ≧1.5×ULN at the ScreeningVisit; 10. Subjects with a positive intradermal skin tuberculin test ≧5mm induration read at 48 to 72 hours after placement; 11. Chestradiograph (at the Screening visit) consistent with prior tuberculosisinfection including, but not limited to, apical scarring, apicalfibrosis, or multiple calcified granulomata. This does not includenon-caseating granulomata; 12. Treatment with oral prednisone orequivalent >10 mg per day within 4 weeks prior to the Screening Visit;13. Use of parenteral or intra-articular glucocorticoids within 4 weeksprior to the Screening Visit; 14. Treatment with anakinra within twoweeks prior to the Screening Visit; 15. Treatment with etanercept,cyclosporine, mycophenolate, taerolimus, gold, penicillamine,sulfasalazine, or hydroxychloroquine within 4 weeks prior to theScreening Visit; 16. Treatment with adalimumab within 6 weeks prior tothe Screening Visit; 17. Treatment with abatacept, azathioprine,cyclophosphamide or infliximab within 12 weeks prior to the ScreeningVisit; 18. Treatment with leflunomide or rituximab within 6 months priorto the Screening Visit; 19. Treatment with tocilizumab or any otheranti-IL-6 medication prior to Screening Visit; 20. Start treatment orchange dose of current treatment with NSAIDs/COX2 inhibitors for 2 weeksprior to Screening; 21. Received administration of any live (attenuated)vaccine within 3 months prior to the Screening Visit; 22. Known historyof Human Immunodeficiency Virus (HIV) antibody; and/or positiveHepatitis B surface antigen (HBsAg), and/or positive Hepatitis Cantibody (HCV) at the Screening Visit; 23. History of malignancy otherthan carcinoma in-situ of the cervix, or adequately treated,non-metastatic squamous or basal cell carcinoma of the skin within fiveyears prior the Screening Visit; 24. History of alcohol or drug abusewithin the 5 years prior to the Screening Visit; 25. Any subject who hashad surgery within 4 weeks prior to the Screening Visit; 26. Anysubjects with planned elective surgery; 27. Participation in anyclinical research study evaluating another investigational drug ortherapy within 30 days or at least 5 half-lives, whichever is longer, ofthe investigational drug, prior to the Screening Visit; 28. Previousexposure to mAb1.

Study Drug Dosage. Subjects had a Screening Visit on Day-14 to Day-3.Once eligibility was confirmed, the subjects were randomly allocated toreceive either mAb1 or placebo. Subjects were enrolled in 4 parallelgroups of 8 subjects each and were dosed with 50, 100 or 200 mg SC mAb1or placebo (1:1:1:1). Each subject received a single SC dose of mAb1 orplacebo on Day 1, and was followed for 6 weeks.

Dose Preparation. Study drug was supplied as lyophilized powder insterile, single-use vials. Each vial contained 269 mg of mAb1 andprovided a stock solution of 100 mg/ml after reconstitution. The maximumdose to be administered per vial was 200 mg. Placebo was supplied inmatched vials. mAb1 was reconstituted in Sterile Water For Injection(WFI), and contained a withdrawable volume of up to 2 ml. The 50 mg dosewas administered at 0.5 ml, 100 mg at 1 ml, 200 mg at 2 ml and theplacebo at 2 ml. All study drug injections were administered in theabdomen.

Screening; Visit 1; Day-14 to Day-3 (±2 days): Informed Consent;Inclusion/Exclusion Criteria; Medical History; Physical Examination;Height and Weight; Vital Signs; Chest x-ray (PA and Lateral); TuberculinSkin Test (read at 48 to 72 hours); hs-CRP and SAA; ESR;Electrocardiogram; Serum βHCG pregnancy test (for women of childbearingpotential); HCV Ab and HBsAg; Hematology panel; Chemistry panel;Urinalysis; Concomitant medications; Adverse Events. Visit 2; BaselineVisit; Day 1: Vital Signs; Weight; Urine pregnancy test (for women ofchildbearing potential); Hematology panel; Chemistry panel; Urinalysis;hs-CRP and SAA; ESR; IL-6; Serum immunoglobulins; RheumatoidFactor/ANA/anti-dsDNA; RNA; Proteomic sample; Pharmacokinetic blooddraw; Anti-mAb1 antibody; Subject's Assessment of Pain; Subject's GlobalAssessment of Disease Activity; Concomitant medications; Adverse events;Randomization; Study drug administration. Visit 3; Day 4: Vital signs;Hematology panel; Chemistry panel; Urinalysis; hs-CRP and SAA; ESR;IL-6; RNA; Proteomic sample; Pharmacokinetic blood draw; Anti-mAb1antibody; Concomitant medications; Adverse events. Visit 4; Day 8: Vitalsigns; Hematology panel; Chemistry panel; Urinalysis; hs-CRP and SAA;ESR; IL-6; RNA; Proteomic sample; Pharmacokinetic blood draw;Concomitant medications; Adverse events. Visit 5: Day 12: Vital signs;Hematology panel; Chemistry panel; Urinalysis; hs-CRP and SAA; ESR;IL-6; RNA; Proteomic sample; Pharmacokinetic blood draw; Concomitantmedications; Adverse events. Visit 6; Day 15: Vital Signs; Urinepregnancy test (for women of childbearing potential); Hematology panel;Chemistry panel; Urinalysis; hs-CRP and SAA; ESR; IL-6; Pharmacokineticblood draw; Concomitant medications; Adverse events. Visit 7: Day 22:Vital signs; Hematology panel; Chemistry panel; Urinalysis; hs-CRP andSAA; ESR; IL-6; RNA; Proteomic sample; Pharmacokinetic blood draw;Concomitant medications; Adverse events. Visit 8; Day 29: Vital Signs;Urine pregnancy test (for women of childbearing potential); Hematologypanel; Chemistry panel; Urinalysis; Electrocardiogram; hs-CRP and SAA;ESR; IL-6; Serum immunoglobulins; Rheumatoid Factor/ANA/anti-dsDNA; RNA;Anti-mAb1 antibody; Proteomic sample; Pharmacokinetic blood draw;Subject's Assessment of Pain; Subject's Global Assessment of DiseaseActivity; Concomitant medications; Adverse events. Visit 9; Day 36:Vital signs; Hematology panel; Chemistry panel; Urinalysis; hs-CRP andSAA; ESR; IL-6; RNA; Proteomic sample; Pharmacokinetic blood draw;Concomitant medications; Adverse events. Visit 10; Day 43; End of Study:Physical examination; Vital signs; Height and weight; Electrocardiogram;Serum βHCG pregnancy test (for women of childbearing potential);Hematology panel; Chemistry panel; Urinalysis; hs-CRP and SAA; ESR;IL-6; Serum immunoglobulins; Rheumatoid Factor/ANA/anti-dsDNA; RNA;Proteomic sample; Pharmacokinetic blood draw; Anti-mAb1 antibody;Subject's Assessment of Pain; Subject's Global Assessment of DiseaseActivity; Concomitant medications; Adverse events.

Results: Baseline levels of RA-associated biomarkers (hsCRP, SAA, ESR,IL-6, Hb, and hepcidin) measured prior to administration of mAb1 orplacebo are shown in Table 14 (n=24).

TABLE 14 hsCRP 21.2 SAA 45713 ESR 48 IL-6 28.8 Hemoglobin 12.3 Meanhepcidin 86.5 Median hepcidin 70.2

The median percent hepcidin change at day 8 is summarized in Table 15.

TABLE 15 mAb1 dose (mg) Study Day 0 50 100 200 D8 −2.4 0.0 −21.0 −66.2

The hepcidin levels for individual study participants at Day 1 and Day 8are set forth in Table 16. UD: Undetected.

TABLE 16 Serum Hepcidin Subject mAb1 (ng/mL) No. Treatment Day 1 Day 81-0008  50 mg 166.2 136.3 3-0003  50 mg 22.7 35.3 4-0003  50 mg 71.927.6 5-0004  50 mg UD UD 5-0006  50 mg 25.0 27.4 6-0001  50 mg 109.2 6.89-0005  50 mg 60.8 — 11-001   50 mg 35.4 37.8 1-0006 100 mg 298.9 203.62-0002 100 mg 97.4 113.7 2-0003 100 mg 70.1 9.0 2-0007 100 mg 189.1 12.43-0002 100 mg UD UD 8-0007 100 mg 38.3 34.4 11-002  100 mg 60.3 20.311-008  100 mg 15.2 18.5 2-0001 200 mg 92.8 11.6 5-0005 200 mg 47.3 20.35-0007 200 mg 12.2 5.2 6-0006 200 mg 12.9 UD 6-0007 200 mg UD UD 10-001 200 mg 171.8 94.2 11-005  200 mg 148.9 37.7 11-007  200 mg 145.1 UD1-0004 Placebo 254.7 242.6 2-0006 Placebo 215.8 73.5 5-0003 Placebo 49.066.0 6-0002 Placebo 75.0 52.1 6-0003 Placebo UD UD 8-0006 Placebo 76.857.1 9-0002 Placebo 133.2 267.6 9-0009 Placebo 47.5 72.1

The percent change in high-sensitivity C-reactive protein (hsCRP) andcirculating IL-6 levels are summarized in Tables 17 and 18,respectively.

TABLE 17 Placebo 50 mg mAb1 100 mg mAb1 200 mg mAb1 Combined Baselinevalue N 8 8 8 8 24 Mean (SD) 29.58 (24.55) 21.97 (16.82) 23.43 (17.53)34.07 (27.46) 26.49 (20.96) Median 21.150 16.250 24.400 29.800 24.200Min:Max 5.43:78.70 5.29:48.20 1.22:44.40 8.12:96.10 1.22:96.10 % Changefrom Baseline at Day 4 N 6 6 4 4 14 Mean (SD) −16.58 (23.84) −7.98(71.17) −39.39 (18.82) −66.54 (15.29) −33.68 (52.26) Median −16.27−21.42 −38.19 −65.24 −46.50 Min:Max −55.4:11.6 −75.1:124.6 −63.3:−17.9−84.5:−51.2 −84.5:124.6 P-value — 0.8182 0.1714 0.0190 0.1093 comparedwith placebo % Change from Baseline at Day 8 N 8 8 8 8 24 Mean (SD) 9.09(52.09) −16.42 (40.23) −69.18 (28.62) −89.21 (6.60) −58.27 (41.70)Median −2.99 −17.27 −72.42 −91.66 −75.97 Min:Max −57.2:95.5 −77.7:36.5−96.6:−8.7 −94.4:−74.3 −96.6:36.5 P-value — 0.4418 0.0011 0.0002 0.0017compared with placebo

TABLE 18 Placebo 50 mg mAb1 100 mg mAb1 200 mg mAb1 Combined Baselinevalue N 8 8 8 8 24 Mean (SD) 33.94 (31.25) 40.39 (26.55) 25.59 (18.58)59.53 (68.35) 41.83 (44.08) Median 21.875 47.160 26.090 24.520 33.400Min:Max 6.22:90.63 5.30:78.42 2.81:45.11 3.69:190.2 2.81:190.02 % Changefrom Baseline at Day 4 N 6 7 4 4 15 Mean (SD) 1.39 (17.49) 514.04 318.17521.69 463.85 (603.32) (191.69) (471.82) (468.89) Median 3.50 244.85259.08 351.40 268.79 Min:Max −25.9:21.2 −45.8:1537.5 158.2:596.3173.2:1210.7 −45.8:1537.5 P-value — 0.0513 0.0095 0.0095 0.0016 comparedwith placebo % Change from Baseline at Day 8 N 8 8 8 8 24 Mean (SD)−8.55 (27.60) −13.21 (59.00) 748.40 966.87 567.69 (685.54) (984.69)(789.60) Median −1.53 −9.10 596.91 647.02 292.18 Min:Max −55.3:24.6−95.4:94.0 31.7:1857.0 −71.4:2836.8 −95.4:2836.8 P-value — 0.7209 0.00020.0104 0.0228 compared with placebo

Median percent changes from baseline for hs-CRP (Table 19), IL-6 (Table20), hemoglobin (Table 21), serum amyloid A (Table 22), and erythrocytesedimentation rate (Table 23) are shown below.

TABLE 19 Placebo 50 mg 100 mg 200 mg Day 1 — — — — Day 4 −16.3 −21.4−38.2 −65.2 Day 8 −3.0 −17.3 −72.4 −91.7 Day 12 −7.7 −9.6 −6.8 −89.0 Day15 16.0 −4.7 −19.8 −82.4 Day 22 −3.7 39.0 −10.8 −32.7 Day 29 1.4 −5.2−38.8 −45.7 Day 36 17.0 −12.3 −28.9 −49.2 Day 43 −19.4 −28.8 14.4 −13.7

TABLE 20 Placebo 50 mg 100 mg 200 mg Day 1 — — — — Day 4 3.5 244.9 259.1351.4 Day 8 −1.5 −9.1 596.1 647.0 Day 12 −43.7 −17.6 69.1 455.6 Day 158.7 −17.6 12.9 36.6 Day 22 −22.6 10.6 −13.8 −12.33 Day 29 16.8 −26.010.3 −28.3 Day 36 −3.7 −18.1 −8.5 −5.9 Day 43 −19.3 −11.6 −8.5 −24.0

TABLE 21 Placebo 50 mg 100 mg 200 mg Day −7 125.5 121 128 127.5 Day 1121.5 125 123.5 121 Day 4 124 112 127 127 Day 8 124 116 126 125.5 Day 12123 120 121.5 122.5 Day 15 123.5 117.5 118 123.5 Day 22 121 120 123 122Day 29 117 115.5 122 124.5 Day 36 118 118 116.5 118 Day 43 121 112.5 120121

TABLE 22 Placebo 50 mg 100 mg 200 mg Day 1 — — — — Day 4 −11.0 −29.1−32.4 −61.9 Day 8 52.2 −14.2 −71.9 −92.5 Day 12 28.8 −38.6 35.2 −92.4Day 15 54.4 −5.6 8.0 −72.7 Day 22 34.6 −9.3 3.3 −40.9 Day 29 41.5 −36.923.3 −32.0 Day 36 23.0 −48.2 −5.0 −34.4 Day 43 10.4 −41.4 19.8 −11.2

TABLE 23 Placebo 50 mg 100 mg 200 mg Day 1 — — — — Day 4 −15 −30 −16.7−6.3 Day 8 −5.6 −31.2 −36.3 −33.8 Day 12 3.0 −26.4 −32.1 −46.8 Day 1518.0 −10.2 −22.9 −36.3 Day 22 12.5 −19.4 −23.1 −18.1 Day 29 26.7 −19.4−28.4 −4.4 Day 36 33.3 −20.7 −33.3 −5.3 Day 43 12.5 −5.2 −24.9 −13.5

Safety was assessed by measuring neutrophils (Table 24) and alanineaminotransferase (ALT) (Table 25).

TABLE 24 Treatment Group Combined Neutrophil 50 mg mAb1 100 mg mAb1 200mg mAb1 mAb1 + MTX Range MTX (n = 8) + MTX (n = 8) + MTX (n = 8) + MTX(n = 8) (n = 24) <1.5 × 10³/μL 0 1 (12.5%) 3 (37.5%) 2 (25%) 6 (25%)<1.0 × 10³/μL 0 0 2 (25%) 0 2 (8.3%) <0.5 × 10³/μL 0 0 0 0 0

TABLE 25 Treatment Group Combined 50 mg mAb1 100 mg mAb1 200 mg mAb1mAb1 + MTX ALT Range MTX (n = 8) + MTX (n = 8) + MTX (n = 8) + MTX (n =8) (n = 24) >1 × ULN 3 (37.5%) 2 (25%) 6 (75%) 5 (62.5%) 13 (54.2%) >2 ×ULN 1 (12.5%) 0 2 (25%) 2 (25%) 4 (16.7%) >3 × ULN 1 (12.5%) 0 0 2 (25%)2 (8.3%) >5 × ULN 0 0 0 1 (12.5%) 1 (4.17%) >8 × ULN 0 0 0 0 0

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A method for treating rheumatoid arthritis (RA) in a subject,comprising: administering to the subject a therapeutically effectiveamount of a human antibody or antigen-binding fragment thereof whichspecifically binds to human interleukin-6 (hIL-6R) with a K_(D) of about500 pM or less as measured by surface plasmon resonance.
 2. The methodof claim 1, wherein the antibody or antigen-binding fragment comprises aCDR sequence combination selected from the group consisting of SEQ IDNO:21/23/25/29/31/33, 149/151/153/157/159/161, 5/7/9/13/15/17 and181/183/185/189/191/193.
 3. The method of claim 1, wherein the antibodyor antigen-binding fragment comprises a HCVR/LCVR pair selected from thegroup consisting of SEQ ID NO:19/27, 147/155, 3/11 and 179/187.
 4. Themethod of claim 1, wherein the antibody or antigen-binding fragmentspecifically binds to human interleukin-6 (hIL-6R) with a K_(D) of about300 pM or less as measured b surface s plasmon resonance.
 5. The methodof claim 1, wherein the human antibody or antigen-binding fragmentcomprises the complementarity determining region (CDR) sequences of aheavy chain variable region (HCVR) and light chain variable region(LCVR) pair (HCVR/LCVR) selected from the group consisting of SEQ IDNO:19/27, 147/155, 3/11 and 179/187.
 6. The method of claim 5, whereinthe antibody or antigen-binding fragment specifically binds to humaninterleukin-6 (hIL-6R) with a K_(D) of about 200 pM or less as measuredby surface plasmon resonance.
 7. The method of claim 5, wherein theantibody or antigen-binding fragment specifically binds to humaninterleukin-6 (hIL-6R) with a K_(D) of about 100 pM or less as measuredby surface plasmon resonance.
 8. The method of claim 7, furthercomprising: a second therapeutic agent selected from the groupconsisting of: methotrexate; sulfasalazine; hydroxychloroquine;leflunomide; etanercept; infliximab; adalimumab; golimumab; rilonacept;anakinra; abatacept; cetiolizumab; and rituximab.
 9. The method of claim1, wherein the therapeutically effective amount of the antibody orantigen-binding fragment is about 50 mg to about 200 mg.
 10. A methodfor treating rheumatoid arthritis in a subject, the method comprising:(a) administering to the subject a first dose of a human antibody orantigen-binding fragment thereof which specifically binds to humaninterleukin-6 receptor (hIL6-R) with a K_(D) of about 500 pM or less asmeasured by surface plasmon resonance, and (b) administering to thesubject at least one subsequent dose of the human antibody orantigen-binding fragment.
 11. The method of claim 10, wherein the humanantibody or antigen-binding fragment comprises the complementaritydetermining region (CDR) sequences of a heavy chain variable region(HCVR) and light chain variable region (LCVR) pair (HCVR/LCVR) selectedfrom the group consisting of SEQ ID NO:147/155, 3/11 and 179/187. 12.The method of claim 10, wherein the first dose and the subsequent doseare each about 50 mg to about 200 mg of the human antibody orantigen-binding fragment.
 13. The method of claim 10, wherein the firstdose and the subsequent dose are administered 1 week to 6 weeks apart.14. The method of claim 12, wherein the first dose and the subsequentdose are each 50 mg, 100 mg, 150 mg or 200 mg and administered 1 weekapart or 2 weeks apart.
 15. The method of claim 14, further comprisingadministering to the subject one or more subsequent doses of the humanantibody or antigen-binding fragment at 50 mg, 100 mg, 150 mg, or 200mg, weekly or biweekly.
 16. The method of claim 10, wherein the humanantibody or antigen-binding fragment comprises wherein the antibody orantigen-binding fragment comprises a CDR sequence combination selectedfrom the group consisting of SEQ ID NO: 149/151/153/157/159/161,5/7/9/13/15/17 and 181/183/185/189/191/193.
 17. The method of claim 16,wherein the antibody or antigen-binding fragment comprises a HCVRcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:147, 3 and
 179. 18. The method of claim 16, wherein theantibody or antigen-binding fragment comprises a LCVR comprising anamino acid sequence selected from the group consisting of SEQ ID NO:155,11 and
 187. 19. The method of claim 16, wherein the antibody orantigen-binding fragment comprises a HCVR/LCVR pair selected from thegroup consisting of SEQ ID NO:147/155, 3/11 and 179/187.
 20. The methodof claim 10, further comprising administering a second therapeutic agentto the subject.